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    Structured Review

    Roche qrt pcr amplification
    cGAS Is Dispensable for the Early Innate Immune Response to Nuclear DNA Damage (A) Immunoblotting analysis of WT and two cGAS −/− HaCaT clones treated with DMSO or 50 μM etoposide for 6 hr. (B and C) WT and cGAS −/− HaCaT cells were treated with DMSO or 50 μM etoposide, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA or 100 ng/mL poly(I:C) for 6 hr before <t>qRT-PCR</t> analysis of IFN-β (B) and IL-6 (C) mRNA expression. (D) IL-6 in supernatants from WT and cGAS −/− HaCaT cells treated with 50 μM etoposide quantified by ELISA. (E) MRC-5 fibroblasts were treated with non-targeting (NT) or cGAS -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide for 6 hr. cGAS protein expression was analyzed by western blot. (F) qRT-PCR analysis of IFN-β mRNA expression in MRC-5 fibroblasts treated with siRNA as in (E) and stimulated with 50 μM etoposide or transfected with 1 μg/mL HT-DNA for 6 hr. (G) PMA-differentiated WT, cGAS −/− , and IFI16 −/− THP1 cells were treated with 50 μM etoposide for 30 hr or 1 μg/mL HT-DNA for 6 hr before qRT-PCR analysis of IFN-β mRNA. (H) WT and cGAS −/− HaCaT cells grown on coverslips were treated with 50 μM etoposide for 4 hr, stained for p65 (green) and DNA (DAPI, blue), and visualized by confocal microscopy. Scale bar, 20 μm. (I) Quantification of p65 translocation from (H). (J) HaCaT cells were treated with 50 μM etoposide for the indicated times or transfected with 1 μg/mL HT-DNA for 4 hr. cGAMP production was quantified by LC-MS. .
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    1) Product Images from "Non-canonical Activation of the DNA Sensing Adaptor STING by ATM and IFI16 Mediates NF-κB Signaling after Nuclear DNA Damage"

    Article Title: Non-canonical Activation of the DNA Sensing Adaptor STING by ATM and IFI16 Mediates NF-κB Signaling after Nuclear DNA Damage

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2018.07.034

    cGAS Is Dispensable for the Early Innate Immune Response to Nuclear DNA Damage (A) Immunoblotting analysis of WT and two cGAS −/− HaCaT clones treated with DMSO or 50 μM etoposide for 6 hr. (B and C) WT and cGAS −/− HaCaT cells were treated with DMSO or 50 μM etoposide, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA or 100 ng/mL poly(I:C) for 6 hr before qRT-PCR analysis of IFN-β (B) and IL-6 (C) mRNA expression. (D) IL-6 in supernatants from WT and cGAS −/− HaCaT cells treated with 50 μM etoposide quantified by ELISA. (E) MRC-5 fibroblasts were treated with non-targeting (NT) or cGAS -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide for 6 hr. cGAS protein expression was analyzed by western blot. (F) qRT-PCR analysis of IFN-β mRNA expression in MRC-5 fibroblasts treated with siRNA as in (E) and stimulated with 50 μM etoposide or transfected with 1 μg/mL HT-DNA for 6 hr. (G) PMA-differentiated WT, cGAS −/− , and IFI16 −/− THP1 cells were treated with 50 μM etoposide for 30 hr or 1 μg/mL HT-DNA for 6 hr before qRT-PCR analysis of IFN-β mRNA. (H) WT and cGAS −/− HaCaT cells grown on coverslips were treated with 50 μM etoposide for 4 hr, stained for p65 (green) and DNA (DAPI, blue), and visualized by confocal microscopy. Scale bar, 20 μm. (I) Quantification of p65 translocation from (H). (J) HaCaT cells were treated with 50 μM etoposide for the indicated times or transfected with 1 μg/mL HT-DNA for 4 hr. cGAMP production was quantified by LC-MS. .
    Figure Legend Snippet: cGAS Is Dispensable for the Early Innate Immune Response to Nuclear DNA Damage (A) Immunoblotting analysis of WT and two cGAS −/− HaCaT clones treated with DMSO or 50 μM etoposide for 6 hr. (B and C) WT and cGAS −/− HaCaT cells were treated with DMSO or 50 μM etoposide, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA or 100 ng/mL poly(I:C) for 6 hr before qRT-PCR analysis of IFN-β (B) and IL-6 (C) mRNA expression. (D) IL-6 in supernatants from WT and cGAS −/− HaCaT cells treated with 50 μM etoposide quantified by ELISA. (E) MRC-5 fibroblasts were treated with non-targeting (NT) or cGAS -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide for 6 hr. cGAS protein expression was analyzed by western blot. (F) qRT-PCR analysis of IFN-β mRNA expression in MRC-5 fibroblasts treated with siRNA as in (E) and stimulated with 50 μM etoposide or transfected with 1 μg/mL HT-DNA for 6 hr. (G) PMA-differentiated WT, cGAS −/− , and IFI16 −/− THP1 cells were treated with 50 μM etoposide for 30 hr or 1 μg/mL HT-DNA for 6 hr before qRT-PCR analysis of IFN-β mRNA. (H) WT and cGAS −/− HaCaT cells grown on coverslips were treated with 50 μM etoposide for 4 hr, stained for p65 (green) and DNA (DAPI, blue), and visualized by confocal microscopy. Scale bar, 20 μm. (I) Quantification of p65 translocation from (H). (J) HaCaT cells were treated with 50 μM etoposide for the indicated times or transfected with 1 μg/mL HT-DNA for 4 hr. cGAMP production was quantified by LC-MS. .

    Techniques Used: Clone Assay, Transfection, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Staining, Confocal Microscopy, Translocation Assay, Liquid Chromatography with Mass Spectroscopy

    Etoposide-Induced NF-κB Activation Involves DNA Damage Factors, but Not TBK1 Activity (A) HaCaT cells grown on coverslips were pre-treated for 30 min with 3 μg/mL brefeldin A where indicated before stimulation with 50 μM etoposide or transfection of 1 μg/mL HT-DNA. Cells were fixed and stained for STING (green) and DNA (DAPI, blue). Scale bar, 20 μm. (B and C) HaCaT cells were pre-treated for 30 min with 3 μg/mL brefeldin A before treatment with 50 μM etoposide or DMSO, mock transfection (Lipo), or transfection of 1 μg/mL HT-DNA for 6 hr. IFN-β (B) and IL-6 (C) mRNA expression was analyzed by qRT-PCR. (D and E) HaCaT cells were pre-treated for 1 hr with 2 μM TBK1 inhibitor MRT67307 and stimulated as in (B) before qRT-PCR analysis of IFN-β (D) and IL-6 (E) mRNA expression. (F) HaCaT cells grown on coverslips were pre-treated with 2 μM TBK1 inhibitor MRT67307 for 1 hr before 4 hr of stimulation with 50 μM etoposide. Cells were fixed and stained for p65 (red) and DNA (DAPI, blue). Scale bar, 20 μm. (G and H) HaCaT cells were pre-treated for 1 hr with 10 μM ATM inhibitor KU55933 and stimulated as in (B). IFN-β (G) and IL-6 (H) mRNA expression was quantified by qRT-PCR. (I) ELISA analysis of IL-6 secretion in supernatants from cells treated as in (G) and stimulated for 24 hr. (J) HaCaT cells grown on coverslips were pre-treated for 1 hr with 10 μM KU55933 before 4 hr of stimulation with 50 μM etoposide. Cells were fixed and stained for p65 (green) and DNA (DAPI, blue). Scale bar, 20 μm. (K) qRT-PCR analysis of IFN-β mRNA expression in NHEK cells pre-treated for 1 hr with 10 μM KU55933, followed by treatment with 50 μM etoposide for 24 hr. (L) qRT-PCR analysis of IFN-β mRNA in HaCaT cells pre-treated for 1 hr with 10 μM PARP inhibitor PJ34 before treatment as in (B) for 6 hr. .
    Figure Legend Snippet: Etoposide-Induced NF-κB Activation Involves DNA Damage Factors, but Not TBK1 Activity (A) HaCaT cells grown on coverslips were pre-treated for 30 min with 3 μg/mL brefeldin A where indicated before stimulation with 50 μM etoposide or transfection of 1 μg/mL HT-DNA. Cells were fixed and stained for STING (green) and DNA (DAPI, blue). Scale bar, 20 μm. (B and C) HaCaT cells were pre-treated for 30 min with 3 μg/mL brefeldin A before treatment with 50 μM etoposide or DMSO, mock transfection (Lipo), or transfection of 1 μg/mL HT-DNA for 6 hr. IFN-β (B) and IL-6 (C) mRNA expression was analyzed by qRT-PCR. (D and E) HaCaT cells were pre-treated for 1 hr with 2 μM TBK1 inhibitor MRT67307 and stimulated as in (B) before qRT-PCR analysis of IFN-β (D) and IL-6 (E) mRNA expression. (F) HaCaT cells grown on coverslips were pre-treated with 2 μM TBK1 inhibitor MRT67307 for 1 hr before 4 hr of stimulation with 50 μM etoposide. Cells were fixed and stained for p65 (red) and DNA (DAPI, blue). Scale bar, 20 μm. (G and H) HaCaT cells were pre-treated for 1 hr with 10 μM ATM inhibitor KU55933 and stimulated as in (B). IFN-β (G) and IL-6 (H) mRNA expression was quantified by qRT-PCR. (I) ELISA analysis of IL-6 secretion in supernatants from cells treated as in (G) and stimulated for 24 hr. (J) HaCaT cells grown on coverslips were pre-treated for 1 hr with 10 μM KU55933 before 4 hr of stimulation with 50 μM etoposide. Cells were fixed and stained for p65 (green) and DNA (DAPI, blue). Scale bar, 20 μm. (K) qRT-PCR analysis of IFN-β mRNA expression in NHEK cells pre-treated for 1 hr with 10 μM KU55933, followed by treatment with 50 μM etoposide for 24 hr. (L) qRT-PCR analysis of IFN-β mRNA in HaCaT cells pre-treated for 1 hr with 10 μM PARP inhibitor PJ34 before treatment as in (B) for 6 hr. .

    Techniques Used: Activation Assay, Activity Assay, Transfection, Staining, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    STING Is Required for the Innate Immune Response to Etoposide-Induced DNA Damage (A) Wild-type (WT) and STING −/− HaCaT cells were treated with DMSO or 50 μM etoposide for 6 hr, and protein expression was analyzed by immunoblotting. (B) Clonogenic survival assay of WT and STING −/− HaCaT cells. Numbers of colonies > 50 cells were counted and expressed as a percentage of untreated control. (C and D) WT HaCaT and two STING −/− clones were treated with DMSO or 50 μM etoposide, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA or 100 ng/mL poly(I:C) for 6 hr before qRT-PCR analysis of IFN-β (C) and IL-6 (D) mRNA expression. (E) ELISA analysis of IL-6 secretion in supernatants from cells treated as in (C) for 24 hr. (F) qRT-PCR array analysis of cytokine and chemokine expression in WT and STING −/− HaCaT cells treated with DMSO, 50 μM etoposide, Lipofectamine, or 1 μg/mL HT-DNA for 6 hr. Shown are genes induced at least 2-fold over controls. (G and H) WT and STING −/− HaCaT cells grown on coverslips were treated with 50 μM etoposide for 4 hr and stained for NF-κB p65 (green) and DNA (DAPI, blue) for analysis by confocal microscopy (G) and quantification of p65 nuclear translocation (H). Scale bar, 20 μm. (I and J) NHEKs were treated with non-targeting (NT) or STING -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide for 24 hr. STING protein levels were analyzed by immunoblotting (I), and IFN-β mRNA expression was quantified by qRT-PCR (J). (K) MRC-5 fibroblasts were treated with non-targeting (NT) or STING -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide for 6 hr and analysis of IFN-β mRNA by RT-PCR. (L) PMA-differentiated WT and STING −/− THP1 cells were stimulated with 50 μM etoposide for 30 hr or 1 μg/mL HT-DNA for 6 hr before qRT-PCR analysis of IFN-β mRNA. A–S3F.
    Figure Legend Snippet: STING Is Required for the Innate Immune Response to Etoposide-Induced DNA Damage (A) Wild-type (WT) and STING −/− HaCaT cells were treated with DMSO or 50 μM etoposide for 6 hr, and protein expression was analyzed by immunoblotting. (B) Clonogenic survival assay of WT and STING −/− HaCaT cells. Numbers of colonies > 50 cells were counted and expressed as a percentage of untreated control. (C and D) WT HaCaT and two STING −/− clones were treated with DMSO or 50 μM etoposide, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA or 100 ng/mL poly(I:C) for 6 hr before qRT-PCR analysis of IFN-β (C) and IL-6 (D) mRNA expression. (E) ELISA analysis of IL-6 secretion in supernatants from cells treated as in (C) for 24 hr. (F) qRT-PCR array analysis of cytokine and chemokine expression in WT and STING −/− HaCaT cells treated with DMSO, 50 μM etoposide, Lipofectamine, or 1 μg/mL HT-DNA for 6 hr. Shown are genes induced at least 2-fold over controls. (G and H) WT and STING −/− HaCaT cells grown on coverslips were treated with 50 μM etoposide for 4 hr and stained for NF-κB p65 (green) and DNA (DAPI, blue) for analysis by confocal microscopy (G) and quantification of p65 nuclear translocation (H). Scale bar, 20 μm. (I and J) NHEKs were treated with non-targeting (NT) or STING -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide for 24 hr. STING protein levels were analyzed by immunoblotting (I), and IFN-β mRNA expression was quantified by qRT-PCR (J). (K) MRC-5 fibroblasts were treated with non-targeting (NT) or STING -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide for 6 hr and analysis of IFN-β mRNA by RT-PCR. (L) PMA-differentiated WT and STING −/− THP1 cells were stimulated with 50 μM etoposide for 30 hr or 1 μg/mL HT-DNA for 6 hr before qRT-PCR analysis of IFN-β mRNA. A–S3F.

    Techniques Used: Expressing, Clonogenic Cell Survival Assay, Clone Assay, Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, Confocal Microscopy, Translocation Assay, Reverse Transcription Polymerase Chain Reaction

    TRAF6 Mediates the K63-Linked Poly-ubiquitylation of STING (A) Immunoprecipitation of TRAF6 and STING from WT and IFI16 −/− HaCaT cells treated with 50 μM etoposide as indicated. Immunoprecipitates (IP) with immunoglobulin G (IgG) control and input lysates were analyzed by immunoblotting. (B) WT and two TRAF6 −/− HaCaT clones were treated with 50 μM etoposide for 6 hr, and protein expression was analyzed by immunoblotting. (C) qRT-PCR analysis of IL-6 mRNA expression in cells treated as in (B). (D) WT and TRAF6 −/− HaCaT cells were treated with 50 μM etoposide or DMSO, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA for 6 hr before qRT-PCR analysis of IFN-β mRNA. (E) Immunoblotting analysis of WT and TRAF6 −/− HaCaT cells treated with 50 μM etoposide for the indicated times. (F) HaCaT cells were pre-treated for 1 hr with the indicated concentrations of Ubc13 inhibitor NSC697923 (NSC) before 6 hr of stimulation with 50 μM etoposide. IL-6 mRNA expression was quantified by qRT-PCR. (G) HEK293T cells were transfected with plasmids for the expression of IFI16, FLAG-tagged TRAF6, and hemagglutinin (HA)-tagged ubiquitin as indicated. 24 hr after transfection, STING was immunoprecipitated, and proteins in immunoprecipitates and input lysates were analyzed by immunoblotting. (H) Immunoprecipitation of K63-linked ubiquitin chains from WT and TRAF6 −/− HaCaT cells treated with 50 μM etoposide for the times indicated. Higher molecular weight forms of modified STING are visualized by gradient SDS-PAGE above the antibody heavy chain ( ∗ ), top panel, together with the association of unmodified STING, lower panel. .
    Figure Legend Snippet: TRAF6 Mediates the K63-Linked Poly-ubiquitylation of STING (A) Immunoprecipitation of TRAF6 and STING from WT and IFI16 −/− HaCaT cells treated with 50 μM etoposide as indicated. Immunoprecipitates (IP) with immunoglobulin G (IgG) control and input lysates were analyzed by immunoblotting. (B) WT and two TRAF6 −/− HaCaT clones were treated with 50 μM etoposide for 6 hr, and protein expression was analyzed by immunoblotting. (C) qRT-PCR analysis of IL-6 mRNA expression in cells treated as in (B). (D) WT and TRAF6 −/− HaCaT cells were treated with 50 μM etoposide or DMSO, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA for 6 hr before qRT-PCR analysis of IFN-β mRNA. (E) Immunoblotting analysis of WT and TRAF6 −/− HaCaT cells treated with 50 μM etoposide for the indicated times. (F) HaCaT cells were pre-treated for 1 hr with the indicated concentrations of Ubc13 inhibitor NSC697923 (NSC) before 6 hr of stimulation with 50 μM etoposide. IL-6 mRNA expression was quantified by qRT-PCR. (G) HEK293T cells were transfected with plasmids for the expression of IFI16, FLAG-tagged TRAF6, and hemagglutinin (HA)-tagged ubiquitin as indicated. 24 hr after transfection, STING was immunoprecipitated, and proteins in immunoprecipitates and input lysates were analyzed by immunoblotting. (H) Immunoprecipitation of K63-linked ubiquitin chains from WT and TRAF6 −/− HaCaT cells treated with 50 μM etoposide for the times indicated. Higher molecular weight forms of modified STING are visualized by gradient SDS-PAGE above the antibody heavy chain ( ∗ ), top panel, together with the association of unmodified STING, lower panel. .

    Techniques Used: Immunoprecipitation, Clone Assay, Expressing, Quantitative RT-PCR, Transfection, Molecular Weight, Modification, SDS Page

    The Innate Immune Response to Etoposide-Induced Damage Involves IFI16 (A) Immunoblotting analysis of WT and IFI16 −/− HaCaT cells stimulated with 50 μM etoposide or DMSO for 6 hr. (B and C) WT HaCaT cells and two IFI16 −/− cell clones were treated for 6 hr with DMSO or 50 μM etoposide, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA or 100 ng/mL poly(I:C). IFN-β (B) or IL-6 (C) mRNA was quantified by qRT-PCR. (D) ELISA analysis of IL-6 protein in supernatants from WT and IFI16 −/− HaCaT cells treated with 50 μM etoposide for indicated times. (E) qRT-PCR analysis of CCL20 mRNA in WT and IFI16 −/− HaCaT cells treated with DMSO or 50 μM etoposide for 6 hr. (F) WT and IFI16 −/− HaCaT cells were treated as in (B) for 4 hr before analysis of protein expression by immunoblotting. (G) WT and IFI16 −/− HaCaT cells grown on coverslips were treated with 50 μM etoposide for 4 hr and fixed and stained for p65 (green) and DNA (DAPI, blue). Scale bar, 20 μm. (H) Quantification of p65 nuclear translocation in cells from (G). (I) Immunoblotting analysis of WT HaCaT cells and IFI16 −/− HaCaT cells reconstituted with lentiviruses for the expression of Luciferase (luc) or IFI16 as indicated. Cells were treated with doxycycline for 24 hr to induce expression and then stimulated with 50 μM etoposide for 6 hr. (J) qRT-PCR analysis of IFN-β mRNA in cells treated as in (I) as indicated. (K–M) MRC-5 fibroblasts treated with non-targeting (NT) or IFI16 -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide or DMSO for 6 hr. IFI16 protein expression was analyzed by immunoblotting (K). IFN-β (L) and IL-6 (M) mRNA levels were analyzed by qRT-PCR. G–S3L.
    Figure Legend Snippet: The Innate Immune Response to Etoposide-Induced Damage Involves IFI16 (A) Immunoblotting analysis of WT and IFI16 −/− HaCaT cells stimulated with 50 μM etoposide or DMSO for 6 hr. (B and C) WT HaCaT cells and two IFI16 −/− cell clones were treated for 6 hr with DMSO or 50 μM etoposide, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA or 100 ng/mL poly(I:C). IFN-β (B) or IL-6 (C) mRNA was quantified by qRT-PCR. (D) ELISA analysis of IL-6 protein in supernatants from WT and IFI16 −/− HaCaT cells treated with 50 μM etoposide for indicated times. (E) qRT-PCR analysis of CCL20 mRNA in WT and IFI16 −/− HaCaT cells treated with DMSO or 50 μM etoposide for 6 hr. (F) WT and IFI16 −/− HaCaT cells were treated as in (B) for 4 hr before analysis of protein expression by immunoblotting. (G) WT and IFI16 −/− HaCaT cells grown on coverslips were treated with 50 μM etoposide for 4 hr and fixed and stained for p65 (green) and DNA (DAPI, blue). Scale bar, 20 μm. (H) Quantification of p65 nuclear translocation in cells from (G). (I) Immunoblotting analysis of WT HaCaT cells and IFI16 −/− HaCaT cells reconstituted with lentiviruses for the expression of Luciferase (luc) or IFI16 as indicated. Cells were treated with doxycycline for 24 hr to induce expression and then stimulated with 50 μM etoposide for 6 hr. (J) qRT-PCR analysis of IFN-β mRNA in cells treated as in (I) as indicated. (K–M) MRC-5 fibroblasts treated with non-targeting (NT) or IFI16 -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide or DMSO for 6 hr. IFI16 protein expression was analyzed by immunoblotting (K). IFN-β (L) and IL-6 (M) mRNA levels were analyzed by qRT-PCR. G–S3L.

    Techniques Used: Clone Assay, Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Staining, Translocation Assay, Luciferase

    Etoposide-Mediated DNA Damage Induces an Acute Innate Immune Response in Human Cells (A–C) HaCaT keratinocytes were treated with 50 μM etoposide for the times indicated before qRT-PCR analysis of IFN-β (A), IL-6 (B), and CCL20 (C) mRNA. (D and E) Supernatants from cells treated with 50 μM etoposide were analyzed for secreted type I IFN using a bio-assay (D) or IL-6 protein using ELISA (E). (F) HaCaT cells were treated with 50 μM etoposide for the times indicated or transfected with 1 μg/mL herring testis (HT)-DNA for 6 hr. Phosphorylation of γH2A.X was analyzed by immunoblotting. (G) Cytotoxicity assay of HaCaT cells treated with 50 μM etoposide for the times indicated or lysed (Lys). (H and I) Primary normal human epidermal keratinocytes (NHEKs) from adult donors were treated with 50 μM etoposide for the times indicated before qRT-PCR analysis of IFN-β (H) and IL-6 (I) mRNA. (J) Supernatants from NHEK cells treated as in (H) were analyzed for IL-6 secretion by ELISA. (K) Cytotoxicity assay of NHEK cells treated as in (H) or lysed (Lys). (L) Primary MRC-5 fibroblasts were treated with 50 μM etoposide before qRT-PCR analysis of IFN-β mRNA expression. (M) Cytotoxicity assay of MRC-5 cells treated with 50 μM etoposide or lysed (Lys). (N) PMA-differentiated THP1 cells were stimulated with 50 μM etoposide for indicated times before qRT-PCR analysis of IFN-β mRNA. (O) Cytotoxicity assay of THP1 cells treated as in (N) or lysed (Lys). .
    Figure Legend Snippet: Etoposide-Mediated DNA Damage Induces an Acute Innate Immune Response in Human Cells (A–C) HaCaT keratinocytes were treated with 50 μM etoposide for the times indicated before qRT-PCR analysis of IFN-β (A), IL-6 (B), and CCL20 (C) mRNA. (D and E) Supernatants from cells treated with 50 μM etoposide were analyzed for secreted type I IFN using a bio-assay (D) or IL-6 protein using ELISA (E). (F) HaCaT cells were treated with 50 μM etoposide for the times indicated or transfected with 1 μg/mL herring testis (HT)-DNA for 6 hr. Phosphorylation of γH2A.X was analyzed by immunoblotting. (G) Cytotoxicity assay of HaCaT cells treated with 50 μM etoposide for the times indicated or lysed (Lys). (H and I) Primary normal human epidermal keratinocytes (NHEKs) from adult donors were treated with 50 μM etoposide for the times indicated before qRT-PCR analysis of IFN-β (H) and IL-6 (I) mRNA. (J) Supernatants from NHEK cells treated as in (H) were analyzed for IL-6 secretion by ELISA. (K) Cytotoxicity assay of NHEK cells treated as in (H) or lysed (Lys). (L) Primary MRC-5 fibroblasts were treated with 50 μM etoposide before qRT-PCR analysis of IFN-β mRNA expression. (M) Cytotoxicity assay of MRC-5 cells treated with 50 μM etoposide or lysed (Lys). (N) PMA-differentiated THP1 cells were stimulated with 50 μM etoposide for indicated times before qRT-PCR analysis of IFN-β mRNA. (O) Cytotoxicity assay of THP1 cells treated as in (N) or lysed (Lys). .

    Techniques Used: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Transfection, Cytotoxicity Assay, Expressing

    Nuclear DNA Damage Results in the Assembly of a Non-canonical Signaling Complex Containing STING (A) Immunoprecipitation of STING from HaCaT cells treated with 50 μM etoposide for the indicated times. Immunoprecipitates (IPs) and whole-cell lysates were analyzed by immunoblotting. (B) Immunoblotting analysis following immunoprecipitation of STING from HaCaT cells treated with 50 μM etoposide or transfected with 1 μg/mL HT-DNA as indicated. (C) Immunoprecipitation of STING from HaCaT cells pre-treated for 1 hr with 10 μM PARP inhibitor PJ34, followed by treatment with 50 μM etoposide for 2 hr. (D) Immunoprecipitation of STING from HaCaT cells pre-treated for 1 hr with 10 μM ATM inhibitor KU55933 followed by treatment with 50 μM etoposide. (E) Immunoprecipitation of STING from WT and IFI16 −/− HaCaT cells treated with 50 μM etoposide as indicated. (F) Immunoprecipitation of IFI16 from WT and STING −/− HaCaT cells treated with 50 μM etoposide as indicated. (G) HEK293T cells transfected with expression constructs for IFI16 and WT p53 or the S15A or S15D p53 mutants as indicated. 24 hr after transfection, IFI16 was immunoprecipitated from lysates. (H) p53 protein levels in HaCaT cells transfected with a non-targeting (NT) or a p53 -targeting siRNA pool for 48 hr before stimulation with 50 μM etoposide for 6 hr. (I) qRT-PCR analysis of IL-6 mRNA expression in cells treated as in (H). .
    Figure Legend Snippet: Nuclear DNA Damage Results in the Assembly of a Non-canonical Signaling Complex Containing STING (A) Immunoprecipitation of STING from HaCaT cells treated with 50 μM etoposide for the indicated times. Immunoprecipitates (IPs) and whole-cell lysates were analyzed by immunoblotting. (B) Immunoblotting analysis following immunoprecipitation of STING from HaCaT cells treated with 50 μM etoposide or transfected with 1 μg/mL HT-DNA as indicated. (C) Immunoprecipitation of STING from HaCaT cells pre-treated for 1 hr with 10 μM PARP inhibitor PJ34, followed by treatment with 50 μM etoposide for 2 hr. (D) Immunoprecipitation of STING from HaCaT cells pre-treated for 1 hr with 10 μM ATM inhibitor KU55933 followed by treatment with 50 μM etoposide. (E) Immunoprecipitation of STING from WT and IFI16 −/− HaCaT cells treated with 50 μM etoposide as indicated. (F) Immunoprecipitation of IFI16 from WT and STING −/− HaCaT cells treated with 50 μM etoposide as indicated. (G) HEK293T cells transfected with expression constructs for IFI16 and WT p53 or the S15A or S15D p53 mutants as indicated. 24 hr after transfection, IFI16 was immunoprecipitated from lysates. (H) p53 protein levels in HaCaT cells transfected with a non-targeting (NT) or a p53 -targeting siRNA pool for 48 hr before stimulation with 50 μM etoposide for 6 hr. (I) qRT-PCR analysis of IL-6 mRNA expression in cells treated as in (H). .

    Techniques Used: Immunoprecipitation, Transfection, Expressing, Construct, Quantitative RT-PCR

    2) Product Images from "Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering"

    Article Title: Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-33242-z

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal human Chondrocytes (hChondrocytes) and ( B ) hChondrocytes undergoing apoptosis or ( C ) both normal and apoptotic chondrocyte cell lines combined.
    Figure Legend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal human Chondrocytes (hChondrocytes) and ( B ) hChondrocytes undergoing apoptosis or ( C ) both normal and apoptotic chondrocyte cell lines combined.

    Techniques Used: Expressing, Quantitative RT-PCR

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm. ( A ) GeNorm results between Control cell groups with normal hChondrocytes, hBMSCs and hADSCs. ( B ) GeNorm results between treated cell groups, i.e. hChondrocytes undergoing apoptosis, chondrogenic differentiated hBMSCs and hADSCs. ( C ) GeNorm results between all untreated and treated cell lines and types.
    Figure Legend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm. ( A ) GeNorm results between Control cell groups with normal hChondrocytes, hBMSCs and hADSCs. ( B ) GeNorm results between treated cell groups, i.e. hChondrocytes undergoing apoptosis, chondrogenic differentiated hBMSCs and hADSCs. ( C ) GeNorm results between all untreated and treated cell lines and types.

    Techniques Used: Expressing, Quantitative RT-PCR

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hBMSCs and ( B ) chondrogenic differentiated hBMSCs separately or ( C ) both cell lines combined.
    Figure Legend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hBMSCs and ( B ) chondrogenic differentiated hBMSCs separately or ( C ) both cell lines combined.

    Techniques Used: Expressing, Quantitative RT-PCR

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) untreated rat rectus abdominis muscle tissue, ( B ) rat rectus abdominis muscle tissue treated with osteogenic medium, and ( C ) both normal and treated rat muscle tissue.
    Figure Legend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) untreated rat rectus abdominis muscle tissue, ( B ) rat rectus abdominis muscle tissue treated with osteogenic medium, and ( C ) both normal and treated rat muscle tissue.

    Techniques Used: Expressing, Quantitative RT-PCR

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hADSCs and ( B ) chondrogenic differentiated hADSCs separately or ( C ) both untreated and treated hADSCs cell lines combined.
    Figure Legend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hADSCs and ( B ) chondrogenic differentiated hADSCs separately or ( C ) both untreated and treated hADSCs cell lines combined.

    Techniques Used: Expressing, Quantitative RT-PCR

    3) Product Images from "Ezrin Mediates Tethering of the γ-Aminobutyric Acid Transporter GAT1 to Actin Filaments Via a C-Terminal PDZ-Interacting Domain"

    Article Title: Ezrin Mediates Tethering of the γ-Aminobutyric Acid Transporter GAT1 to Actin Filaments Via a C-Terminal PDZ-Interacting Domain

    Journal: Biophysical Journal

    doi: 10.1016/j.bpj.2008.11.070

    Determination of expression of ezrin and Pals1 in N2a cells by qRT-PCR, and an interaction between ezrin and GAT1-YFP8 by FRET. ( A ) mRNA levels of β -actin, γ -actin, ezrin, and Pals1, normalized to β -actin expression. One-step qRT-PCR shows that ezrin is expressed in N2a cells at levels similar to those of PDZ protein Pals1. ( B – D ) FRET results. Prebleaching and postbleaching images of respective CFP and YFP fused proteins. Images are processed using Image J software with the enhance-image plugin (Rasband, W.S.; US National Institutes of Health, Bethesda, MD). Scale bar, 5 μ m. ( E ) GAT1-YFP8 and ezrin-CFP interact, as represented by the 25% ± 3% increase in ezrin-CFP fluorescence that accompanies photodestruction of GAT1-YFP8. The disruption of actin through the addition of 5 μ m latrunculin B significantly decreased FRET between ezrin-CFP and GAT1-YFP8. As a positive control, FRET between YFP-ezrin and ezrin-CFP was performed, resulting in a 26% ± 3% increase in ezrin-CFP fluorescence that accompanied the photodestruction of YFP-ezrin. ( F ) FRET efficiency for ezrin-CFP/GAT1-YFP8 is 19% ± 2%, for ezrin-CFP/GAT1-YFP8 + latrunculin B it is 7% ± 5%, and for ezrin-CFP/YFP-ezrin it is 20% ± 2%. Values are represented as the mean ± SE of 23 replicates. Significance was determined by one-way analysis of variance with Tukey's post hoc test ( p
    Figure Legend Snippet: Determination of expression of ezrin and Pals1 in N2a cells by qRT-PCR, and an interaction between ezrin and GAT1-YFP8 by FRET. ( A ) mRNA levels of β -actin, γ -actin, ezrin, and Pals1, normalized to β -actin expression. One-step qRT-PCR shows that ezrin is expressed in N2a cells at levels similar to those of PDZ protein Pals1. ( B – D ) FRET results. Prebleaching and postbleaching images of respective CFP and YFP fused proteins. Images are processed using Image J software with the enhance-image plugin (Rasband, W.S.; US National Institutes of Health, Bethesda, MD). Scale bar, 5 μ m. ( E ) GAT1-YFP8 and ezrin-CFP interact, as represented by the 25% ± 3% increase in ezrin-CFP fluorescence that accompanies photodestruction of GAT1-YFP8. The disruption of actin through the addition of 5 μ m latrunculin B significantly decreased FRET between ezrin-CFP and GAT1-YFP8. As a positive control, FRET between YFP-ezrin and ezrin-CFP was performed, resulting in a 26% ± 3% increase in ezrin-CFP fluorescence that accompanied the photodestruction of YFP-ezrin. ( F ) FRET efficiency for ezrin-CFP/GAT1-YFP8 is 19% ± 2%, for ezrin-CFP/GAT1-YFP8 + latrunculin B it is 7% ± 5%, and for ezrin-CFP/YFP-ezrin it is 20% ± 2%. Values are represented as the mean ± SE of 23 replicates. Significance was determined by one-way analysis of variance with Tukey's post hoc test ( p

    Techniques Used: Expressing, Quantitative RT-PCR, Software, Fluorescence, Positive Control

    4) Product Images from "New Role for DCR-1/Dicer in Caenorhabditis elegans Innate Immunity against the Highly Virulent Bacterium Bacillus thuringiensis DB27"

    Article Title: New Role for DCR-1/Dicer in Caenorhabditis elegans Innate Immunity against the Highly Virulent Bacterium Bacillus thuringiensis DB27

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00700-13

    Transcriptional response of nasp-1 mutant worms to B. thuringiensis DB27 infection. (A) qRT-PCR confirmation of microarray results. Wild-type (wt) and nasp-1 mutant worms were exposed to B. thuringiensis DB27 for 4 h and used for RNA isolation. The data
    Figure Legend Snippet: Transcriptional response of nasp-1 mutant worms to B. thuringiensis DB27 infection. (A) qRT-PCR confirmation of microarray results. Wild-type (wt) and nasp-1 mutant worms were exposed to B. thuringiensis DB27 for 4 h and used for RNA isolation. The data

    Techniques Used: Mutagenesis, Infection, Quantitative RT-PCR, Microarray, Isolation

    Collagen gene col-92 acts downstream of nasp-1 and is required for resistance to B. thuringiensis ) show high expression in dcr-1 mutant worms as determined by qRT-PCR. The
    Figure Legend Snippet: Collagen gene col-92 acts downstream of nasp-1 and is required for resistance to B. thuringiensis ) show high expression in dcr-1 mutant worms as determined by qRT-PCR. The

    Techniques Used: Expressing, Mutagenesis, Quantitative RT-PCR

    5) Product Images from "Direct and site-specific quantification of RNA 2′-O-methylation by PCR with an engineered DNA polymerase"

    Article Title: Direct and site-specific quantification of RNA 2′-O-methylation by PCR with an engineered DNA polymerase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw200

    Rational design of RT-KTQ-LSIM libraries. Amino acids in immediate proximity to the 2′-oxygen of the nucleotide paired to the incoming dNTP were selected for saturation mutagenesis (namely G668, V669, G672, R746, K747 and N750). Adapted from PDB 4BWM ( 24 ) using PyMOL (Schrödinger, LLC, New York, NY, USA).
    Figure Legend Snippet: Rational design of RT-KTQ-LSIM libraries. Amino acids in immediate proximity to the 2′-oxygen of the nucleotide paired to the incoming dNTP were selected for saturation mutagenesis (namely G668, V669, G672, R746, K747 and N750). Adapted from PDB 4BWM ( 24 ) using PyMOL (Schrödinger, LLC, New York, NY, USA).

    Techniques Used: Mutagenesis

    DNA synthesis catalyzed by RT-KTQ-LSIM is hampered by 2′-O-methylation of RNA templates. ( A ) Structures of relevant nucleotides. ( B ) Primer extension in presence of methylated or unmethylated RNA templates catalyzed by RT-KTQ-LSIM. ( C ) qRT-PCR amplification of methylated and unmethylated RNA oligonucleotides catalyzed by RT-KTQ-LSIM.
    Figure Legend Snippet: DNA synthesis catalyzed by RT-KTQ-LSIM is hampered by 2′-O-methylation of RNA templates. ( A ) Structures of relevant nucleotides. ( B ) Primer extension in presence of methylated or unmethylated RNA templates catalyzed by RT-KTQ-LSIM. ( C ) qRT-PCR amplification of methylated and unmethylated RNA oligonucleotides catalyzed by RT-KTQ-LSIM.

    Techniques Used: DNA Synthesis, Methylation, Quantitative RT-PCR, Amplification

    RT-KTQ-LSIM V669L features increased discrimination between 2′-O-methylated and unmethlyated RNA templates and enables quantification of 2′-O-methylation by qRT-PCR. ( A ) Primer extension in the presence of methylated or unmethlyated RNA templates catalyzed by RT-KTQ-LSIM V669L. ( B ) qRT-PCR amplification of methylated and unmethylated RNA oligonucleotides catalyzed by RT-KTQ-LSIM V669L. ( C ) RT-PCR reactions were stopped after 25 cycles (top) or 30 cycles (bottom) and analyzed by agarose gel electrophoresis. ( D ) The ΔC T -method was used to calculate methylation ratio of RNA template at 100 pM concentration with varied fractions of 2′OmeA/A at the target position. Error bars describe SD (n = 3).
    Figure Legend Snippet: RT-KTQ-LSIM V669L features increased discrimination between 2′-O-methylated and unmethlyated RNA templates and enables quantification of 2′-O-methylation by qRT-PCR. ( A ) Primer extension in the presence of methylated or unmethlyated RNA templates catalyzed by RT-KTQ-LSIM V669L. ( B ) qRT-PCR amplification of methylated and unmethylated RNA oligonucleotides catalyzed by RT-KTQ-LSIM V669L. ( C ) RT-PCR reactions were stopped after 25 cycles (top) or 30 cycles (bottom) and analyzed by agarose gel electrophoresis. ( D ) The ΔC T -method was used to calculate methylation ratio of RNA template at 100 pM concentration with varied fractions of 2′OmeA/A at the target position. Error bars describe SD (n = 3).

    Techniques Used: Methylation, Quantitative RT-PCR, Amplification, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Concentration Assay

    6) Product Images from "Chemoprobe-based assays of histone lysine demethylase 1A target occupation enable in vivo pharmacokinetics and pharmacodynamics studies of KDM1A inhibitors"

    Article Title: Chemoprobe-based assays of histone lysine demethylase 1A target occupation enable in vivo pharmacokinetics and pharmacodynamics studies of KDM1A inhibitors

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.006980

    Analysis of KDM1A target engagement and proxy markers. A , IC 50 determination of KDM1A target engagement by ORY-1001 in MV(4;11) cells treated for 96 h with different doses of ORY-1001 (representative luminescent immunoassay of N = 3, n = 3, mean ± S.D.). B , EC 50 determination of H3K4me2 accumulation induced in MV(4;11) cells by 96 h of treatment with ORY-1001 (AlphaLISA; N = 3, n = 2, mean ± S.E. ( error bars )). C , EC 50 determination of MV(4;11) viability (Alamar Blue assay; representative experiment of N = 5, n = 3, mean ± S.D. ( error bars )). D , EC 50 determination of LY96 gene expression changes induced in MV(4;11) cells by 96 h of treatment with ORY-1001 (qRT-PCR; N = 3, n = 3, mean ± S.E. ( error bars )). Data are normalized to the GAPDH control, and -fold changes were calculated relative to the vehicle control sample.
    Figure Legend Snippet: Analysis of KDM1A target engagement and proxy markers. A , IC 50 determination of KDM1A target engagement by ORY-1001 in MV(4;11) cells treated for 96 h with different doses of ORY-1001 (representative luminescent immunoassay of N = 3, n = 3, mean ± S.D.). B , EC 50 determination of H3K4me2 accumulation induced in MV(4;11) cells by 96 h of treatment with ORY-1001 (AlphaLISA; N = 3, n = 2, mean ± S.E. ( error bars )). C , EC 50 determination of MV(4;11) viability (Alamar Blue assay; representative experiment of N = 5, n = 3, mean ± S.D. ( error bars )). D , EC 50 determination of LY96 gene expression changes induced in MV(4;11) cells by 96 h of treatment with ORY-1001 (qRT-PCR; N = 3, n = 3, mean ± S.E. ( error bars )). Data are normalized to the GAPDH control, and -fold changes were calculated relative to the vehicle control sample.

    Techniques Used: Alamar Blue Assay, Expressing, Quantitative RT-PCR

    7) Product Images from "Epithelial cell lysates induce ExoS expression and secretion by Pseudomonas aeruginosa"

    Article Title: Epithelial cell lysates induce ExoS expression and secretion by Pseudomonas aeruginosa

    Journal: FEMS Microbiology Letters

    doi: 10.1093/femsle/fny053

    Western immunoblots of P. aeruginosa culture supernatants (Sup) and bacterial cell pellets (Pel) after growth for 16 h in TSB, corneal cell lysates or TSBi ( A ) P. aeruginosa PAO1 growth in corneal cell lysates resulted in the appearance of a protein in the culture supernatant recognized by anti-ExoS antibody. The protein was not associated with bacterial cell pellets. Growth in TSBi, but not TSB, resulted in detection of both secreted and bacterial cell-associated ExoS and ExoT. ( B ) Growth of P. aeruginosa PAO1 in heat-treated cell lysates (55°C 1 h, 100°C, 5 min) resulted in the continued appearance of the protein in culture supernatants. Gel loading was normalized to the number of bacteria present.
    Figure Legend Snippet: Western immunoblots of P. aeruginosa culture supernatants (Sup) and bacterial cell pellets (Pel) after growth for 16 h in TSB, corneal cell lysates or TSBi ( A ) P. aeruginosa PAO1 growth in corneal cell lysates resulted in the appearance of a protein in the culture supernatant recognized by anti-ExoS antibody. The protein was not associated with bacterial cell pellets. Growth in TSBi, but not TSB, resulted in detection of both secreted and bacterial cell-associated ExoS and ExoT. ( B ) Growth of P. aeruginosa PAO1 in heat-treated cell lysates (55°C 1 h, 100°C, 5 min) resulted in the continued appearance of the protein in culture supernatants. Gel loading was normalized to the number of bacteria present.

    Techniques Used: Western Blot

    8) Product Images from "Molecular Features and Expression Patterns of Vitellogenin Receptor in Calliptamus italicus (Orthoptera: Acrididae)"

    Article Title: Molecular Features and Expression Patterns of Vitellogenin Receptor in Calliptamus italicus (Orthoptera: Acrididae)

    Journal: Journal of Insect Science

    doi: 10.1093/jisesa/iez119

    Expression pattern of CiVgR in different developmental stages. The total RNA was extracted from the ovarian tissues of the fourth- and fifth-instar nymph of Calliptamus italicus (n4, n5), as well as 1- to 25-d-old female Calliptamus italicus after adult emergence (1–25); β-actin was used as the internal reference. The data were described with means ± standard divisions ( n = 3). The different letters indicate the statistically significant differences ( P
    Figure Legend Snippet: Expression pattern of CiVgR in different developmental stages. The total RNA was extracted from the ovarian tissues of the fourth- and fifth-instar nymph of Calliptamus italicus (n4, n5), as well as 1- to 25-d-old female Calliptamus italicus after adult emergence (1–25); β-actin was used as the internal reference. The data were described with means ± standard divisions ( n = 3). The different letters indicate the statistically significant differences ( P

    Techniques Used: Expressing

    Alignment of the amino acid sequences of VgR between Calliptamus italicus and other insects. Black arrows indicate LDLRa; black dotted lines indicate LDLRb; red boxes show the YWTD motifs; black box indicates the TMD; and black double lines show the CD. C. italicus : VgR from Calliptamus italicus (QBM78333); P. americana : VgR from Periplaneta americana (BAC02725); C. secundus : VgR from Cryptotermes secundus (XP_023710287); Z. nevadensis : VgR from Zootermopsis nevadensis (XP_021934248); F. occidentalis : VgR from Frankliniella occidentalis (XP_026271484); B. germanica : VgR from Blattella germanica (CAJ19121).
    Figure Legend Snippet: Alignment of the amino acid sequences of VgR between Calliptamus italicus and other insects. Black arrows indicate LDLRa; black dotted lines indicate LDLRb; red boxes show the YWTD motifs; black box indicates the TMD; and black double lines show the CD. C. italicus : VgR from Calliptamus italicus (QBM78333); P. americana : VgR from Periplaneta americana (BAC02725); C. secundus : VgR from Cryptotermes secundus (XP_023710287); Z. nevadensis : VgR from Zootermopsis nevadensis (XP_021934248); F. occidentalis : VgR from Frankliniella occidentalis (XP_026271484); B. germanica : VgR from Blattella germanica (CAJ19121).

    Techniques Used:

    9) Product Images from "Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering"

    Article Title: Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-33242-z

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm. ( A ) GeNorm results between Control cell groups with normal hChondrocytes, hBMSCs and hADSCs. ( B ) GeNorm results between treated cell groups, i.e. hChondrocytes undergoing apoptosis, chondrogenic differentiated hBMSCs and hADSCs. ( C ) GeNorm results between all untreated and treated cell lines and types.
    Figure Legend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm. ( A ) GeNorm results between Control cell groups with normal hChondrocytes, hBMSCs and hADSCs. ( B ) GeNorm results between treated cell groups, i.e. hChondrocytes undergoing apoptosis, chondrogenic differentiated hBMSCs and hADSCs. ( C ) GeNorm results between all untreated and treated cell lines and types.

    Techniques Used: Expressing, Quantitative RT-PCR

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hADSCs and ( B ) chondrogenic differentiated hADSCs separately or ( C ) both untreated and treated hADSCs cell lines combined.
    Figure Legend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hADSCs and ( B ) chondrogenic differentiated hADSCs separately or ( C ) both untreated and treated hADSCs cell lines combined.

    Techniques Used: Expressing, Quantitative RT-PCR

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal human Chondrocytes (hChondrocytes) and ( B ) hChondrocytes undergoing apoptosis or ( C ) both normal and apoptotic chondrocyte cell lines combined.
    Figure Legend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal human Chondrocytes (hChondrocytes) and ( B ) hChondrocytes undergoing apoptosis or ( C ) both normal and apoptotic chondrocyte cell lines combined.

    Techniques Used: Expressing, Quantitative RT-PCR

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hBMSCs and ( B ) chondrogenic differentiated hBMSCs separately or ( C ) both cell lines combined.
    Figure Legend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hBMSCs and ( B ) chondrogenic differentiated hBMSCs separately or ( C ) both cell lines combined.

    Techniques Used: Expressing, Quantitative RT-PCR

    10) Product Images from "Dacomitinib, a pan-inhibitor of ErbB receptors, suppresses growth and invasive capacity of chemoresistant ovarian carcinoma cells"

    Article Title: Dacomitinib, a pan-inhibitor of ErbB receptors, suppresses growth and invasive capacity of chemoresistant ovarian carcinoma cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-04147-0

    Dacomitinib inhibits PLK1-FOXM1 pathway. ( A ) HRG/HER3 loop activates PLK1. The effects of HRGβ-1 on PLK1 activation was determined by Western blot analysis. Protein lysates from serum-starved and HRGβ-1-treated cells were subjected to Western blotting and probed with the indicated antibodies. The blots are representative of three independent experiments with similar outcomes. ( B ) PLK1 blockade increases cisplatin sensitivity. The effects of BI 2536-cisplatin therapy on cell proliferation were investigated by MTT assay and shown by IC 50 shift analysis. The cultures were treated with BI 2536 (20 nM) and cisplatin (0.1, 0.5, 1, 2.5, 5 and 10 μg/mL) for 48 h. ( C ) Normalised isobolograms of combination of BI 2536 and cisplatin. ( D ) The effects of cetuximab (10 μg/mL), trastuzumab (10 μg/mL), H3.105.5 (10 μg/mL), erlotinib (5 μM) and dacomitinib (5 μM) on PLK1-FOXM1 pathway and its down-stream targets were determined by Western blot analysis. The blots are representative of three independent experiments with similar outcomes. ( E ) The effects of the anti-ErbB agents on the expression of PLK1-FOXM1 targets genes were determined by qRT-PCR analysis. Data shown represent the mean ± SD from three independent experiments, each performed in triplicate. Statistically significant values of * p
    Figure Legend Snippet: Dacomitinib inhibits PLK1-FOXM1 pathway. ( A ) HRG/HER3 loop activates PLK1. The effects of HRGβ-1 on PLK1 activation was determined by Western blot analysis. Protein lysates from serum-starved and HRGβ-1-treated cells were subjected to Western blotting and probed with the indicated antibodies. The blots are representative of three independent experiments with similar outcomes. ( B ) PLK1 blockade increases cisplatin sensitivity. The effects of BI 2536-cisplatin therapy on cell proliferation were investigated by MTT assay and shown by IC 50 shift analysis. The cultures were treated with BI 2536 (20 nM) and cisplatin (0.1, 0.5, 1, 2.5, 5 and 10 μg/mL) for 48 h. ( C ) Normalised isobolograms of combination of BI 2536 and cisplatin. ( D ) The effects of cetuximab (10 μg/mL), trastuzumab (10 μg/mL), H3.105.5 (10 μg/mL), erlotinib (5 μM) and dacomitinib (5 μM) on PLK1-FOXM1 pathway and its down-stream targets were determined by Western blot analysis. The blots are representative of three independent experiments with similar outcomes. ( E ) The effects of the anti-ErbB agents on the expression of PLK1-FOXM1 targets genes were determined by qRT-PCR analysis. Data shown represent the mean ± SD from three independent experiments, each performed in triplicate. Statistically significant values of * p

    Techniques Used: Activation Assay, Western Blot, MTT Assay, Expressing, Quantitative RT-PCR

    Dacomitinib inhibits migration and invasion. ( A ) The effects of the anti-ErbB agents on expression of EMT markers were determined by qRT-PCR analysis. ( B ) The effects of the ErbB inhibitors on cell migration and invasion. The cells were placed into 8-μm porous culture inserts, treated with the drugs and allowed to migrate for 48 h. The migrated cells on the lower surface of the inserts were quantified by crystal violet staining. For invasion assay, the cells were placed into matrigel-coated inserts and allowed to invade through the matrigel layer for 48 h. Data shown represent the mean ± SD from three independent experiments, each performed in triplicate. Statistically significant values of * p
    Figure Legend Snippet: Dacomitinib inhibits migration and invasion. ( A ) The effects of the anti-ErbB agents on expression of EMT markers were determined by qRT-PCR analysis. ( B ) The effects of the ErbB inhibitors on cell migration and invasion. The cells were placed into 8-μm porous culture inserts, treated with the drugs and allowed to migrate for 48 h. The migrated cells on the lower surface of the inserts were quantified by crystal violet staining. For invasion assay, the cells were placed into matrigel-coated inserts and allowed to invade through the matrigel layer for 48 h. Data shown represent the mean ± SD from three independent experiments, each performed in triplicate. Statistically significant values of * p

    Techniques Used: Migration, Expressing, Quantitative RT-PCR, Staining, Invasion Assay

    Expression of the ErbB family in the EOC cells. ( A , B ) The mRNA levels of the ErbB family in the EOC cell lines were determined by qRT-PCR. The data were evaluated in triplicate and collected from three independent experiments. Gene expression levels were normalised to B2M in each cell line. Data were analysed by one-way ANOVA followed by Tukey’s post hoc test and are shown as mean ± SD. Statistically significant values of * p
    Figure Legend Snippet: Expression of the ErbB family in the EOC cells. ( A , B ) The mRNA levels of the ErbB family in the EOC cell lines were determined by qRT-PCR. The data were evaluated in triplicate and collected from three independent experiments. Gene expression levels were normalised to B2M in each cell line. Data were analysed by one-way ANOVA followed by Tukey’s post hoc test and are shown as mean ± SD. Statistically significant values of * p

    Techniques Used: Expressing, Quantitative RT-PCR

    11) Product Images from "Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering"

    Article Title: Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-33242-z

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal human Chondrocytes (hChondrocytes) and ( B ) hChondrocytes undergoing apoptosis or ( C ) both normal and apoptotic chondrocyte cell lines combined.
    Figure Legend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal human Chondrocytes (hChondrocytes) and ( B ) hChondrocytes undergoing apoptosis or ( C ) both normal and apoptotic chondrocyte cell lines combined.

    Techniques Used: Expressing, Quantitative RT-PCR

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm. ( A ) GeNorm results between Control cell groups with normal hChondrocytes, hBMSCs and hADSCs. ( B ) GeNorm results between treated cell groups, i.e. hChondrocytes undergoing apoptosis, chondrogenic differentiated hBMSCs and hADSCs. ( C ) GeNorm results between all untreated and treated cell lines and types.
    Figure Legend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm. ( A ) GeNorm results between Control cell groups with normal hChondrocytes, hBMSCs and hADSCs. ( B ) GeNorm results between treated cell groups, i.e. hChondrocytes undergoing apoptosis, chondrogenic differentiated hBMSCs and hADSCs. ( C ) GeNorm results between all untreated and treated cell lines and types.

    Techniques Used: Expressing, Quantitative RT-PCR

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hBMSCs and ( B ) chondrogenic differentiated hBMSCs separately or ( C ) both cell lines combined.
    Figure Legend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hBMSCs and ( B ) chondrogenic differentiated hBMSCs separately or ( C ) both cell lines combined.

    Techniques Used: Expressing, Quantitative RT-PCR

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) untreated rat rectus abdominis muscle tissue, ( B ) rat rectus abdominis muscle tissue treated with osteogenic medium, and ( C ) both normal and treated rat muscle tissue.
    Figure Legend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) untreated rat rectus abdominis muscle tissue, ( B ) rat rectus abdominis muscle tissue treated with osteogenic medium, and ( C ) both normal and treated rat muscle tissue.

    Techniques Used: Expressing, Quantitative RT-PCR

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hADSCs and ( B ) chondrogenic differentiated hADSCs separately or ( C ) both untreated and treated hADSCs cell lines combined.
    Figure Legend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hADSCs and ( B ) chondrogenic differentiated hADSCs separately or ( C ) both untreated and treated hADSCs cell lines combined.

    Techniques Used: Expressing, Quantitative RT-PCR

    12) Product Images from "Blockade of vascular endothelial growth factor receptors by tivozanib has potential anti-tumour effects on human glioblastoma cells"

    Article Title: Blockade of vascular endothelial growth factor receptors by tivozanib has potential anti-tumour effects on human glioblastoma cells

    Journal: Scientific Reports

    doi: 10.1038/srep44075

    Tivozanib induces G2/M cell cycle arrest. ( A ) Following treatment with tivozanib for 48 h, the cell pellets were fixed and incubated with PI to analyse the cell cycle distribution on a flow cytometer. The graphs are representative of three independent experiments with similar results. ( B ) The cells were treated with tivozanib for 48 h then total RNA was harvested for qRT-PCR analysis. ( C ) Protein lysates from tivozanib-treated cells were subjected to Western blotting and probed with the indicated antibodies. β-actin was used as the loading control. The blots are representative of three independent experiments with similar outcomes. Data are given as mean ± SD. Statistically significant values of * p
    Figure Legend Snippet: Tivozanib induces G2/M cell cycle arrest. ( A ) Following treatment with tivozanib for 48 h, the cell pellets were fixed and incubated with PI to analyse the cell cycle distribution on a flow cytometer. The graphs are representative of three independent experiments with similar results. ( B ) The cells were treated with tivozanib for 48 h then total RNA was harvested for qRT-PCR analysis. ( C ) Protein lysates from tivozanib-treated cells were subjected to Western blotting and probed with the indicated antibodies. β-actin was used as the loading control. The blots are representative of three independent experiments with similar outcomes. Data are given as mean ± SD. Statistically significant values of * p

    Techniques Used: Incubation, Flow Cytometry, Cytometry, Quantitative RT-PCR, Western Blot

    Tivozanib inhibits adhesive and invasive potential of the GBM cells. ( A ) Tivozanib-treated cells were seeded into collagen I-coated culture dishes then the adhesive cells were stained, lysed and the optical densitometry was read. ( B,C ) The effects of tivozanib on mRNA and protein levels of ICAM-1 and VCAM-1 were measured by qRT-PCR and Western blot analysis. β-actin was used as the loading control. The blots are representative of three independent experiments with similar results. ( D ) Equal amounts of secreted protein from treated cells were incubated with a synthetic substrate labelled with amino-4-trifluoromethyl coumarin (AFC). The substrate is cleaved by cathepsin B to release AFC, which is fluorometrically detected. ( E ) The conditioned media from each sample was subjected to a chromogenic substrate, which is cleaved by active uPA and produces a colorimetrically detectable product. (F) The conditioned media was collected and separated on a non-reducing polyacrylamide gel containing gelatin A. Gelatinolytic activities are visualized as clear bands against the blue background of stained gelatin. The zymograms are representative of three independent experiments with similar results. The gels were cropped and the full-length gels are presented in Supplementary Fig. 3 . (G) The cells were placed into 8-μm porous culture inserts, treated with tivozanib and allowed to migrate for 48 h. The migrated cells on the lower surface of the inserts were fixed with methanol, stained with crystal violet, lysed with 30% acetic acid and the optical densitometry was measured at 590 nm. (H) For invasion assay, the cells were placed into matrigel-coated inserts and allowed to invade through the matrigel layer for 48 h. Data are given as mean ± SD. Statistically significant values of * p
    Figure Legend Snippet: Tivozanib inhibits adhesive and invasive potential of the GBM cells. ( A ) Tivozanib-treated cells were seeded into collagen I-coated culture dishes then the adhesive cells were stained, lysed and the optical densitometry was read. ( B,C ) The effects of tivozanib on mRNA and protein levels of ICAM-1 and VCAM-1 were measured by qRT-PCR and Western blot analysis. β-actin was used as the loading control. The blots are representative of three independent experiments with similar results. ( D ) Equal amounts of secreted protein from treated cells were incubated with a synthetic substrate labelled with amino-4-trifluoromethyl coumarin (AFC). The substrate is cleaved by cathepsin B to release AFC, which is fluorometrically detected. ( E ) The conditioned media from each sample was subjected to a chromogenic substrate, which is cleaved by active uPA and produces a colorimetrically detectable product. (F) The conditioned media was collected and separated on a non-reducing polyacrylamide gel containing gelatin A. Gelatinolytic activities are visualized as clear bands against the blue background of stained gelatin. The zymograms are representative of three independent experiments with similar results. The gels were cropped and the full-length gels are presented in Supplementary Fig. 3 . (G) The cells were placed into 8-μm porous culture inserts, treated with tivozanib and allowed to migrate for 48 h. The migrated cells on the lower surface of the inserts were fixed with methanol, stained with crystal violet, lysed with 30% acetic acid and the optical densitometry was measured at 590 nm. (H) For invasion assay, the cells were placed into matrigel-coated inserts and allowed to invade through the matrigel layer for 48 h. Data are given as mean ± SD. Statistically significant values of * p

    Techniques Used: Staining, Quantitative RT-PCR, Western Blot, Incubation, Invasion Assay

    13) Product Images from "Non-canonical Activation of the DNA Sensing Adaptor STING by ATM and IFI16 Mediates NF-κB Signaling after Nuclear DNA Damage"

    Article Title: Non-canonical Activation of the DNA Sensing Adaptor STING by ATM and IFI16 Mediates NF-κB Signaling after Nuclear DNA Damage

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2018.07.034

    cGAS Is Dispensable for the Early Innate Immune Response to Nuclear DNA Damage (A) Immunoblotting analysis of WT and two cGAS −/− HaCaT clones treated with DMSO or 50 μM etoposide for 6 hr. (B and C) WT and cGAS −/− HaCaT cells were treated with DMSO or 50 μM etoposide, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA or 100 ng/mL poly(I:C) for 6 hr before qRT-PCR analysis of IFN-β (B) and IL-6 (C) mRNA expression. (D) IL-6 in supernatants from WT and cGAS −/− HaCaT cells treated with 50 μM etoposide quantified by ELISA. (E) MRC-5 fibroblasts were treated with non-targeting (NT) or cGAS -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide for 6 hr. cGAS protein expression was analyzed by western blot. (F) qRT-PCR analysis of IFN-β mRNA expression in MRC-5 fibroblasts treated with siRNA as in (E) and stimulated with 50 μM etoposide or transfected with 1 μg/mL HT-DNA for 6 hr. (G) PMA-differentiated WT, cGAS −/− , and IFI16 −/− THP1 cells were treated with 50 μM etoposide for 30 hr or 1 μg/mL HT-DNA for 6 hr before qRT-PCR analysis of IFN-β mRNA. (H) WT and cGAS −/− HaCaT cells grown on coverslips were treated with 50 μM etoposide for 4 hr, stained for p65 (green) and DNA (DAPI, blue), and visualized by confocal microscopy. Scale bar, 20 μm. (I) Quantification of p65 translocation from (H). (J) HaCaT cells were treated with 50 μM etoposide for the indicated times or transfected with 1 μg/mL HT-DNA for 4 hr. cGAMP production was quantified by LC-MS. Data are presented as mean values of biological triplicates ± SD. See also Figure S4 .
    Figure Legend Snippet: cGAS Is Dispensable for the Early Innate Immune Response to Nuclear DNA Damage (A) Immunoblotting analysis of WT and two cGAS −/− HaCaT clones treated with DMSO or 50 μM etoposide for 6 hr. (B and C) WT and cGAS −/− HaCaT cells were treated with DMSO or 50 μM etoposide, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA or 100 ng/mL poly(I:C) for 6 hr before qRT-PCR analysis of IFN-β (B) and IL-6 (C) mRNA expression. (D) IL-6 in supernatants from WT and cGAS −/− HaCaT cells treated with 50 μM etoposide quantified by ELISA. (E) MRC-5 fibroblasts were treated with non-targeting (NT) or cGAS -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide for 6 hr. cGAS protein expression was analyzed by western blot. (F) qRT-PCR analysis of IFN-β mRNA expression in MRC-5 fibroblasts treated with siRNA as in (E) and stimulated with 50 μM etoposide or transfected with 1 μg/mL HT-DNA for 6 hr. (G) PMA-differentiated WT, cGAS −/− , and IFI16 −/− THP1 cells were treated with 50 μM etoposide for 30 hr or 1 μg/mL HT-DNA for 6 hr before qRT-PCR analysis of IFN-β mRNA. (H) WT and cGAS −/− HaCaT cells grown on coverslips were treated with 50 μM etoposide for 4 hr, stained for p65 (green) and DNA (DAPI, blue), and visualized by confocal microscopy. Scale bar, 20 μm. (I) Quantification of p65 translocation from (H). (J) HaCaT cells were treated with 50 μM etoposide for the indicated times or transfected with 1 μg/mL HT-DNA for 4 hr. cGAMP production was quantified by LC-MS. Data are presented as mean values of biological triplicates ± SD. See also Figure S4 .

    Techniques Used: Clone Assay, Transfection, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Staining, Confocal Microscopy, Translocation Assay, Liquid Chromatography with Mass Spectroscopy

    Etoposide-Induced NF-κB Activation Involves DNA Damage Factors, but Not TBK1 Activity (A) HaCaT cells grown on coverslips were pre-treated for 30 min with 3 μg/mL brefeldin A where indicated before stimulation with 50 μM etoposide or transfection of 1 μg/mL HT-DNA. Cells were fixed and stained for STING (green) and DNA (DAPI, blue). Scale bar, 20 μm. (B and C) HaCaT cells were pre-treated for 30 min with 3 μg/mL brefeldin A before treatment with 50 μM etoposide or DMSO, mock transfection (Lipo), or transfection of 1 μg/mL HT-DNA for 6 hr. IFN-β (B) and IL-6 (C) mRNA expression was analyzed by qRT-PCR. (D and E) HaCaT cells were pre-treated for 1 hr with 2 μM TBK1 inhibitor MRT67307 and stimulated as in (B) before qRT-PCR analysis of IFN-β (D) and IL-6 (E) mRNA expression. (F) HaCaT cells grown on coverslips were pre-treated with 2 μM TBK1 inhibitor MRT67307 for 1 hr before 4 hr of stimulation with 50 μM etoposide. Cells were fixed and stained for p65 (red) and DNA (DAPI, blue). Scale bar, 20 μm. (G and H) HaCaT cells were pre-treated for 1 hr with 10 μM ATM inhibitor KU55933 and stimulated as in (B). IFN-β (G) and IL-6 (H) mRNA expression was quantified by qRT-PCR. (I) ELISA analysis of IL-6 secretion in supernatants from cells treated as in (G) and stimulated for 24 hr. (J) HaCaT cells grown on coverslips were pre-treated for 1 hr with 10 μM KU55933 before 4 hr of stimulation with 50 μM etoposide. Cells were fixed and stained for p65 (green) and DNA (DAPI, blue). Scale bar, 20 μm. (K) qRT-PCR analysis of IFN-β mRNA expression in NHEK cells pre-treated for 1 hr with 10 μM KU55933, followed by treatment with 50 μM etoposide for 24 hr. (L) qRT-PCR analysis of IFN-β mRNA in HaCaT cells pre-treated for 1 hr with 10 μM PARP inhibitor PJ34 before treatment as in (B) for 6 hr. Data are presented as mean values of biological triplicates ± SD. See also Figure S5 .
    Figure Legend Snippet: Etoposide-Induced NF-κB Activation Involves DNA Damage Factors, but Not TBK1 Activity (A) HaCaT cells grown on coverslips were pre-treated for 30 min with 3 μg/mL brefeldin A where indicated before stimulation with 50 μM etoposide or transfection of 1 μg/mL HT-DNA. Cells were fixed and stained for STING (green) and DNA (DAPI, blue). Scale bar, 20 μm. (B and C) HaCaT cells were pre-treated for 30 min with 3 μg/mL brefeldin A before treatment with 50 μM etoposide or DMSO, mock transfection (Lipo), or transfection of 1 μg/mL HT-DNA for 6 hr. IFN-β (B) and IL-6 (C) mRNA expression was analyzed by qRT-PCR. (D and E) HaCaT cells were pre-treated for 1 hr with 2 μM TBK1 inhibitor MRT67307 and stimulated as in (B) before qRT-PCR analysis of IFN-β (D) and IL-6 (E) mRNA expression. (F) HaCaT cells grown on coverslips were pre-treated with 2 μM TBK1 inhibitor MRT67307 for 1 hr before 4 hr of stimulation with 50 μM etoposide. Cells were fixed and stained for p65 (red) and DNA (DAPI, blue). Scale bar, 20 μm. (G and H) HaCaT cells were pre-treated for 1 hr with 10 μM ATM inhibitor KU55933 and stimulated as in (B). IFN-β (G) and IL-6 (H) mRNA expression was quantified by qRT-PCR. (I) ELISA analysis of IL-6 secretion in supernatants from cells treated as in (G) and stimulated for 24 hr. (J) HaCaT cells grown on coverslips were pre-treated for 1 hr with 10 μM KU55933 before 4 hr of stimulation with 50 μM etoposide. Cells were fixed and stained for p65 (green) and DNA (DAPI, blue). Scale bar, 20 μm. (K) qRT-PCR analysis of IFN-β mRNA expression in NHEK cells pre-treated for 1 hr with 10 μM KU55933, followed by treatment with 50 μM etoposide for 24 hr. (L) qRT-PCR analysis of IFN-β mRNA in HaCaT cells pre-treated for 1 hr with 10 μM PARP inhibitor PJ34 before treatment as in (B) for 6 hr. Data are presented as mean values of biological triplicates ± SD. See also Figure S5 .

    Techniques Used: Activation Assay, Activity Assay, Transfection, Staining, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    STING Is Required for the Innate Immune Response to Etoposide-Induced DNA Damage (A) Wild-type (WT) and STING −/− HaCaT cells were treated with DMSO or 50 μM etoposide for 6 hr, and protein expression was analyzed by immunoblotting. (B) Clonogenic survival assay of WT and STING −/− HaCaT cells. Numbers of colonies > 50 cells were counted and expressed as a percentage of untreated control. (C and D) WT HaCaT and two STING −/− clones were treated with DMSO or 50 μM etoposide, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA or 100 ng/mL poly(I:C) for 6 hr before qRT-PCR analysis of IFN-β (C) and IL-6 (D) mRNA expression. (E) ELISA analysis of IL-6 secretion in supernatants from cells treated as in (C) for 24 hr. (F) qRT-PCR array analysis of cytokine and chemokine expression in WT and STING −/− HaCaT cells treated with DMSO, 50 μM etoposide, Lipofectamine, or 1 μg/mL HT-DNA for 6 hr. Shown are genes induced at least 2-fold over controls. (G and H) WT and STING −/− HaCaT cells grown on coverslips were treated with 50 μM etoposide for 4 hr and stained for NF-κB p65 (green) and DNA (DAPI, blue) for analysis by confocal microscopy (G) and quantification of p65 nuclear translocation (H). Scale bar, 20 μm. (I and J) NHEKs were treated with non-targeting (NT) or STING -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide for 24 hr. STING protein levels were analyzed by immunoblotting (I), and IFN-β mRNA expression was quantified by qRT-PCR (J). (K) MRC-5 fibroblasts were treated with non-targeting (NT) or STING -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide for 6 hr and analysis of IFN-β mRNA by RT-PCR. (L) PMA-differentiated WT and STING −/− THP1 cells were stimulated with 50 μM etoposide for 30 hr or 1 μg/mL HT-DNA for 6 hr before qRT-PCR analysis of IFN-β mRNA. Data are presented as mean values of biological triplicates ± SD. See also Figures S2 and S3 A–S3F.
    Figure Legend Snippet: STING Is Required for the Innate Immune Response to Etoposide-Induced DNA Damage (A) Wild-type (WT) and STING −/− HaCaT cells were treated with DMSO or 50 μM etoposide for 6 hr, and protein expression was analyzed by immunoblotting. (B) Clonogenic survival assay of WT and STING −/− HaCaT cells. Numbers of colonies > 50 cells were counted and expressed as a percentage of untreated control. (C and D) WT HaCaT and two STING −/− clones were treated with DMSO or 50 μM etoposide, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA or 100 ng/mL poly(I:C) for 6 hr before qRT-PCR analysis of IFN-β (C) and IL-6 (D) mRNA expression. (E) ELISA analysis of IL-6 secretion in supernatants from cells treated as in (C) for 24 hr. (F) qRT-PCR array analysis of cytokine and chemokine expression in WT and STING −/− HaCaT cells treated with DMSO, 50 μM etoposide, Lipofectamine, or 1 μg/mL HT-DNA for 6 hr. Shown are genes induced at least 2-fold over controls. (G and H) WT and STING −/− HaCaT cells grown on coverslips were treated with 50 μM etoposide for 4 hr and stained for NF-κB p65 (green) and DNA (DAPI, blue) for analysis by confocal microscopy (G) and quantification of p65 nuclear translocation (H). Scale bar, 20 μm. (I and J) NHEKs were treated with non-targeting (NT) or STING -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide for 24 hr. STING protein levels were analyzed by immunoblotting (I), and IFN-β mRNA expression was quantified by qRT-PCR (J). (K) MRC-5 fibroblasts were treated with non-targeting (NT) or STING -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide for 6 hr and analysis of IFN-β mRNA by RT-PCR. (L) PMA-differentiated WT and STING −/− THP1 cells were stimulated with 50 μM etoposide for 30 hr or 1 μg/mL HT-DNA for 6 hr before qRT-PCR analysis of IFN-β mRNA. Data are presented as mean values of biological triplicates ± SD. See also Figures S2 and S3 A–S3F.

    Techniques Used: Expressing, Clonogenic Cell Survival Assay, Clone Assay, Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, Confocal Microscopy, Translocation Assay, Reverse Transcription Polymerase Chain Reaction

    TRAF6 Mediates the K63-Linked Poly-ubiquitylation of STING (A) Immunoprecipitation of TRAF6 and STING from WT and IFI16 −/− HaCaT cells treated with 50 μM etoposide as indicated. Immunoprecipitates (IP) with immunoglobulin G (IgG) control and input lysates were analyzed by immunoblotting. (B) WT and two TRAF6 −/− HaCaT clones were treated with 50 μM etoposide for 6 hr, and protein expression was analyzed by immunoblotting. (C) qRT-PCR analysis of IL-6 mRNA expression in cells treated as in (B). (D) WT and TRAF6 −/− HaCaT cells were treated with 50 μM etoposide or DMSO, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA for 6 hr before qRT-PCR analysis of IFN-β mRNA. (E) Immunoblotting analysis of WT and TRAF6 −/− HaCaT cells treated with 50 μM etoposide for the indicated times. (F) HaCaT cells were pre-treated for 1 hr with the indicated concentrations of Ubc13 inhibitor NSC697923 (NSC) before 6 hr of stimulation with 50 μM etoposide. IL-6 mRNA expression was quantified by qRT-PCR. (G) HEK293T cells were transfected with plasmids for the expression of IFI16, FLAG-tagged TRAF6, and hemagglutinin (HA)-tagged ubiquitin as indicated. 24 hr after transfection, STING was immunoprecipitated, and proteins in immunoprecipitates and input lysates were analyzed by immunoblotting. (H) Immunoprecipitation of K63-linked ubiquitin chains from WT and TRAF6 −/− HaCaT cells treated with 50 μM etoposide for the times indicated. Higher molecular weight forms of modified STING are visualized by gradient SDS-PAGE above the antibody heavy chain ( ∗ ), top panel, together with the association of unmodified STING, lower panel. See also Figure S7 .
    Figure Legend Snippet: TRAF6 Mediates the K63-Linked Poly-ubiquitylation of STING (A) Immunoprecipitation of TRAF6 and STING from WT and IFI16 −/− HaCaT cells treated with 50 μM etoposide as indicated. Immunoprecipitates (IP) with immunoglobulin G (IgG) control and input lysates were analyzed by immunoblotting. (B) WT and two TRAF6 −/− HaCaT clones were treated with 50 μM etoposide for 6 hr, and protein expression was analyzed by immunoblotting. (C) qRT-PCR analysis of IL-6 mRNA expression in cells treated as in (B). (D) WT and TRAF6 −/− HaCaT cells were treated with 50 μM etoposide or DMSO, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA for 6 hr before qRT-PCR analysis of IFN-β mRNA. (E) Immunoblotting analysis of WT and TRAF6 −/− HaCaT cells treated with 50 μM etoposide for the indicated times. (F) HaCaT cells were pre-treated for 1 hr with the indicated concentrations of Ubc13 inhibitor NSC697923 (NSC) before 6 hr of stimulation with 50 μM etoposide. IL-6 mRNA expression was quantified by qRT-PCR. (G) HEK293T cells were transfected with plasmids for the expression of IFI16, FLAG-tagged TRAF6, and hemagglutinin (HA)-tagged ubiquitin as indicated. 24 hr after transfection, STING was immunoprecipitated, and proteins in immunoprecipitates and input lysates were analyzed by immunoblotting. (H) Immunoprecipitation of K63-linked ubiquitin chains from WT and TRAF6 −/− HaCaT cells treated with 50 μM etoposide for the times indicated. Higher molecular weight forms of modified STING are visualized by gradient SDS-PAGE above the antibody heavy chain ( ∗ ), top panel, together with the association of unmodified STING, lower panel. See also Figure S7 .

    Techniques Used: Immunoprecipitation, Clone Assay, Expressing, Quantitative RT-PCR, Transfection, Molecular Weight, Modification, SDS Page

    The Innate Immune Response to Etoposide-Induced Damage Involves IFI16 (A) Immunoblotting analysis of WT and IFI16 −/− HaCaT cells stimulated with 50 μM etoposide or DMSO for 6 hr. (B and C) WT HaCaT cells and two IFI16 −/− cell clones were treated for 6 hr with DMSO or 50 μM etoposide, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA or 100 ng/mL poly(I:C). IFN-β (B) or IL-6 (C) mRNA was quantified by qRT-PCR. (D) ELISA analysis of IL-6 protein in supernatants from WT and IFI16 −/− HaCaT cells treated with 50 μM etoposide for indicated times. (E) qRT-PCR analysis of CCL20 mRNA in WT and IFI16 −/− HaCaT cells treated with DMSO or 50 μM etoposide for 6 hr. (F) WT and IFI16 −/− HaCaT cells were treated as in (B) for 4 hr before analysis of protein expression by immunoblotting. (G) WT and IFI16 −/− HaCaT cells grown on coverslips were treated with 50 μM etoposide for 4 hr and fixed and stained for p65 (green) and DNA (DAPI, blue). Scale bar, 20 μm. (H) Quantification of p65 nuclear translocation in cells from (G). (I) Immunoblotting analysis of WT HaCaT cells and IFI16 −/− HaCaT cells reconstituted with lentiviruses for the expression of Luciferase (luc) or IFI16 as indicated. Cells were treated with doxycycline for 24 hr to induce expression and then stimulated with 50 μM etoposide for 6 hr. (J) qRT-PCR analysis of IFN-β mRNA in cells treated as in (I) as indicated. (K–M) MRC-5 fibroblasts treated with non-targeting (NT) or IFI16 -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide or DMSO for 6 hr. IFI16 protein expression was analyzed by immunoblotting (K). IFN-β (L) and IL-6 (M) mRNA levels were analyzed by qRT-PCR. Data are presented as mean values of biological triplicates ± SD. See also Figures S3 G–S3L.
    Figure Legend Snippet: The Innate Immune Response to Etoposide-Induced Damage Involves IFI16 (A) Immunoblotting analysis of WT and IFI16 −/− HaCaT cells stimulated with 50 μM etoposide or DMSO for 6 hr. (B and C) WT HaCaT cells and two IFI16 −/− cell clones were treated for 6 hr with DMSO or 50 μM etoposide, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA or 100 ng/mL poly(I:C). IFN-β (B) or IL-6 (C) mRNA was quantified by qRT-PCR. (D) ELISA analysis of IL-6 protein in supernatants from WT and IFI16 −/− HaCaT cells treated with 50 μM etoposide for indicated times. (E) qRT-PCR analysis of CCL20 mRNA in WT and IFI16 −/− HaCaT cells treated with DMSO or 50 μM etoposide for 6 hr. (F) WT and IFI16 −/− HaCaT cells were treated as in (B) for 4 hr before analysis of protein expression by immunoblotting. (G) WT and IFI16 −/− HaCaT cells grown on coverslips were treated with 50 μM etoposide for 4 hr and fixed and stained for p65 (green) and DNA (DAPI, blue). Scale bar, 20 μm. (H) Quantification of p65 nuclear translocation in cells from (G). (I) Immunoblotting analysis of WT HaCaT cells and IFI16 −/− HaCaT cells reconstituted with lentiviruses for the expression of Luciferase (luc) or IFI16 as indicated. Cells were treated with doxycycline for 24 hr to induce expression and then stimulated with 50 μM etoposide for 6 hr. (J) qRT-PCR analysis of IFN-β mRNA in cells treated as in (I) as indicated. (K–M) MRC-5 fibroblasts treated with non-targeting (NT) or IFI16 -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide or DMSO for 6 hr. IFI16 protein expression was analyzed by immunoblotting (K). IFN-β (L) and IL-6 (M) mRNA levels were analyzed by qRT-PCR. Data are presented as mean values of biological triplicates ± SD. See also Figures S3 G–S3L.

    Techniques Used: Clone Assay, Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Staining, Translocation Assay, Luciferase

    Etoposide-Mediated DNA Damage Induces an Acute Innate Immune Response in Human Cells (A–C) HaCaT keratinocytes were treated with 50 μM etoposide for the times indicated before qRT-PCR analysis of IFN-β (A), IL-6 (B), and CCL20 (C) mRNA. (D and E) Supernatants from cells treated with 50 μM etoposide were analyzed for secreted type I IFN using a bio-assay (D) or IL-6 protein using ELISA (E). (F) HaCaT cells were treated with 50 μM etoposide for the times indicated or transfected with 1 μg/mL herring testis (HT)-DNA for 6 hr. Phosphorylation of γH2A.X was analyzed by immunoblotting. (G) Cytotoxicity assay of HaCaT cells treated with 50 μM etoposide for the times indicated or lysed (Lys). (H and I) Primary normal human epidermal keratinocytes (NHEKs) from adult donors were treated with 50 μM etoposide for the times indicated before qRT-PCR analysis of IFN-β (H) and IL-6 (I) mRNA. (J) Supernatants from NHEK cells treated as in (H) were analyzed for IL-6 secretion by ELISA. (K) Cytotoxicity assay of NHEK cells treated as in (H) or lysed (Lys). (L) Primary MRC-5 fibroblasts were treated with 50 μM etoposide before qRT-PCR analysis of IFN-β mRNA expression. (M) Cytotoxicity assay of MRC-5 cells treated with 50 μM etoposide or lysed (Lys). (N) PMA-differentiated THP1 cells were stimulated with 50 μM etoposide for indicated times before qRT-PCR analysis of IFN-β mRNA. (O) Cytotoxicity assay of THP1 cells treated as in (N) or lysed (Lys). Data are presented as mean values of biological triplicates ± SD. See also Figure S1 .
    Figure Legend Snippet: Etoposide-Mediated DNA Damage Induces an Acute Innate Immune Response in Human Cells (A–C) HaCaT keratinocytes were treated with 50 μM etoposide for the times indicated before qRT-PCR analysis of IFN-β (A), IL-6 (B), and CCL20 (C) mRNA. (D and E) Supernatants from cells treated with 50 μM etoposide were analyzed for secreted type I IFN using a bio-assay (D) or IL-6 protein using ELISA (E). (F) HaCaT cells were treated with 50 μM etoposide for the times indicated or transfected with 1 μg/mL herring testis (HT)-DNA for 6 hr. Phosphorylation of γH2A.X was analyzed by immunoblotting. (G) Cytotoxicity assay of HaCaT cells treated with 50 μM etoposide for the times indicated or lysed (Lys). (H and I) Primary normal human epidermal keratinocytes (NHEKs) from adult donors were treated with 50 μM etoposide for the times indicated before qRT-PCR analysis of IFN-β (H) and IL-6 (I) mRNA. (J) Supernatants from NHEK cells treated as in (H) were analyzed for IL-6 secretion by ELISA. (K) Cytotoxicity assay of NHEK cells treated as in (H) or lysed (Lys). (L) Primary MRC-5 fibroblasts were treated with 50 μM etoposide before qRT-PCR analysis of IFN-β mRNA expression. (M) Cytotoxicity assay of MRC-5 cells treated with 50 μM etoposide or lysed (Lys). (N) PMA-differentiated THP1 cells were stimulated with 50 μM etoposide for indicated times before qRT-PCR analysis of IFN-β mRNA. (O) Cytotoxicity assay of THP1 cells treated as in (N) or lysed (Lys). Data are presented as mean values of biological triplicates ± SD. See also Figure S1 .

    Techniques Used: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Transfection, Cytotoxicity Assay, Expressing

    Nuclear DNA Damage Results in the Assembly of a Non-canonical Signaling Complex Containing STING (A) Immunoprecipitation of STING from HaCaT cells treated with 50 μM etoposide for the indicated times. Immunoprecipitates (IPs) and whole-cell lysates were analyzed by immunoblotting. (B) Immunoblotting analysis following immunoprecipitation of STING from HaCaT cells treated with 50 μM etoposide or transfected with 1 μg/mL HT-DNA as indicated. (C) Immunoprecipitation of STING from HaCaT cells pre-treated for 1 hr with 10 μM PARP inhibitor PJ34, followed by treatment with 50 μM etoposide for 2 hr. (D) Immunoprecipitation of STING from HaCaT cells pre-treated for 1 hr with 10 μM ATM inhibitor KU55933 followed by treatment with 50 μM etoposide. (E) Immunoprecipitation of STING from WT and IFI16 −/− HaCaT cells treated with 50 μM etoposide as indicated. (F) Immunoprecipitation of IFI16 from WT and STING −/− HaCaT cells treated with 50 μM etoposide as indicated. (G) HEK293T cells transfected with expression constructs for IFI16 and WT p53 or the S15A or S15D p53 mutants as indicated. 24 hr after transfection, IFI16 was immunoprecipitated from lysates. (H) p53 protein levels in HaCaT cells transfected with a non-targeting (NT) or a p53 -targeting siRNA pool for 48 hr before stimulation with 50 μM etoposide for 6 hr. (I) qRT-PCR analysis of IL-6 mRNA expression in cells treated as in (H). See also Figure S6 .
    Figure Legend Snippet: Nuclear DNA Damage Results in the Assembly of a Non-canonical Signaling Complex Containing STING (A) Immunoprecipitation of STING from HaCaT cells treated with 50 μM etoposide for the indicated times. Immunoprecipitates (IPs) and whole-cell lysates were analyzed by immunoblotting. (B) Immunoblotting analysis following immunoprecipitation of STING from HaCaT cells treated with 50 μM etoposide or transfected with 1 μg/mL HT-DNA as indicated. (C) Immunoprecipitation of STING from HaCaT cells pre-treated for 1 hr with 10 μM PARP inhibitor PJ34, followed by treatment with 50 μM etoposide for 2 hr. (D) Immunoprecipitation of STING from HaCaT cells pre-treated for 1 hr with 10 μM ATM inhibitor KU55933 followed by treatment with 50 μM etoposide. (E) Immunoprecipitation of STING from WT and IFI16 −/− HaCaT cells treated with 50 μM etoposide as indicated. (F) Immunoprecipitation of IFI16 from WT and STING −/− HaCaT cells treated with 50 μM etoposide as indicated. (G) HEK293T cells transfected with expression constructs for IFI16 and WT p53 or the S15A or S15D p53 mutants as indicated. 24 hr after transfection, IFI16 was immunoprecipitated from lysates. (H) p53 protein levels in HaCaT cells transfected with a non-targeting (NT) or a p53 -targeting siRNA pool for 48 hr before stimulation with 50 μM etoposide for 6 hr. (I) qRT-PCR analysis of IL-6 mRNA expression in cells treated as in (H). See also Figure S6 .

    Techniques Used: Immunoprecipitation, Transfection, Expressing, Construct, Quantitative RT-PCR

    14) Product Images from "Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering"

    Article Title: Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-33242-z

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm. ( A ) GeNorm results between Control cell groups with normal hChondrocytes, hBMSCs and hADSCs. ( B ) GeNorm results between treated cell groups, i.e. hChondrocytes undergoing apoptosis, chondrogenic differentiated hBMSCs and hADSCs. ( C ) GeNorm results between all untreated and treated cell lines and types.
    Figure Legend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm. ( A ) GeNorm results between Control cell groups with normal hChondrocytes, hBMSCs and hADSCs. ( B ) GeNorm results between treated cell groups, i.e. hChondrocytes undergoing apoptosis, chondrogenic differentiated hBMSCs and hADSCs. ( C ) GeNorm results between all untreated and treated cell lines and types.

    Techniques Used: Expressing, Quantitative RT-PCR

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hADSCs and ( B ) chondrogenic differentiated hADSCs separately or ( C ) both untreated and treated hADSCs cell lines combined.
    Figure Legend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hADSCs and ( B ) chondrogenic differentiated hADSCs separately or ( C ) both untreated and treated hADSCs cell lines combined.

    Techniques Used: Expressing, Quantitative RT-PCR

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal human Chondrocytes (hChondrocytes) and ( B ) hChondrocytes undergoing apoptosis or ( C ) both normal and apoptotic chondrocyte cell lines combined.
    Figure Legend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal human Chondrocytes (hChondrocytes) and ( B ) hChondrocytes undergoing apoptosis or ( C ) both normal and apoptotic chondrocyte cell lines combined.

    Techniques Used: Expressing, Quantitative RT-PCR

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hBMSCs and ( B ) chondrogenic differentiated hBMSCs separately or ( C ) both cell lines combined.
    Figure Legend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hBMSCs and ( B ) chondrogenic differentiated hBMSCs separately or ( C ) both cell lines combined.

    Techniques Used: Expressing, Quantitative RT-PCR

    15) Product Images from "MicroRNA-96 Promotes Schistosomiasis Hepatic Fibrosis in Mice by Suppressing Smad7"

    Article Title: MicroRNA-96 Promotes Schistosomiasis Hepatic Fibrosis in Mice by Suppressing Smad7

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1016/j.omtm.2018.10.002

    TGF-β1 Elevates miR-96 Expression through Post-transcriptional Regulation (A) qRT-PCR analysis of miR-96 expression in isolated hepatocytes, HSCs, and Kupffer cells from mouse livers at 0 and 50 dpi, and the miR-96 expression in HSCs of the wild-type mice at 0 and 50 dpi. (B) qRT-PCR analysis of tgf-β1 and il13 expression in the HSCs of infected mice. (C) qRT-PCR analysis of miR-96 and miR-214 expression in primary HSCs treated in vitro for 24 hr with IL10 (25 ng/mL), interferon (IFN)-γ (25 ng/mL), TGF-β1 (100 ng/mL), and IL13 (200 ng/mL). (D) qRT-PCR analysis of miR-96 expression in primary HSCs treated with different concentrations of TGF-β1 as indicated. (E) qRT-PCR analysis of miR-96 expression in primary HSCs treated with TGF-β1 (8 ng/mL) for various times. (F) qRT-PCR analysis of pri-miR-96 and pre-miR-96 expression levels in isolated HSCs from mice at 0 and 50 dpi. (G) qRT-PCR analysis of pri-miR-96 and pre-miR-96 expression in primary HSCs treated with TGF-β1 (10 ng/mL) for various times. (H) RNA-binding protein immunoprecipitation (RIP) analysis of post-transcriptional processing of miR-96 . The cell lysates of HSCs isolated from infected (50 dpi) and uninfected (0 dpi) mice were immunoprecipitated with anti-SMAD2+SMAD3, anti-DROSHA, or normal rabbit IgG and subjected to qRT-PCR analysis. (I) In vitro RIP analysis of post-transcriptional processing of miR-96 and miR-21 maturation. qRT-PCR analysis of miR-96 and miR-21 expression in primary HSCs treated with TGF-β1 (100 ng/mL) or IL13 (200 ng/mL) for 24 hr. HSC lysates were immunoprecipitated and subjected to qRT-PCR analysis as described above. Data are represented as mean ± SD. *p
    Figure Legend Snippet: TGF-β1 Elevates miR-96 Expression through Post-transcriptional Regulation (A) qRT-PCR analysis of miR-96 expression in isolated hepatocytes, HSCs, and Kupffer cells from mouse livers at 0 and 50 dpi, and the miR-96 expression in HSCs of the wild-type mice at 0 and 50 dpi. (B) qRT-PCR analysis of tgf-β1 and il13 expression in the HSCs of infected mice. (C) qRT-PCR analysis of miR-96 and miR-214 expression in primary HSCs treated in vitro for 24 hr with IL10 (25 ng/mL), interferon (IFN)-γ (25 ng/mL), TGF-β1 (100 ng/mL), and IL13 (200 ng/mL). (D) qRT-PCR analysis of miR-96 expression in primary HSCs treated with different concentrations of TGF-β1 as indicated. (E) qRT-PCR analysis of miR-96 expression in primary HSCs treated with TGF-β1 (8 ng/mL) for various times. (F) qRT-PCR analysis of pri-miR-96 and pre-miR-96 expression levels in isolated HSCs from mice at 0 and 50 dpi. (G) qRT-PCR analysis of pri-miR-96 and pre-miR-96 expression in primary HSCs treated with TGF-β1 (10 ng/mL) for various times. (H) RNA-binding protein immunoprecipitation (RIP) analysis of post-transcriptional processing of miR-96 . The cell lysates of HSCs isolated from infected (50 dpi) and uninfected (0 dpi) mice were immunoprecipitated with anti-SMAD2+SMAD3, anti-DROSHA, or normal rabbit IgG and subjected to qRT-PCR analysis. (I) In vitro RIP analysis of post-transcriptional processing of miR-96 and miR-21 maturation. qRT-PCR analysis of miR-96 and miR-21 expression in primary HSCs treated with TGF-β1 (100 ng/mL) or IL13 (200 ng/mL) for 24 hr. HSC lysates were immunoprecipitated and subjected to qRT-PCR analysis as described above. Data are represented as mean ± SD. *p

    Techniques Used: Expressing, Quantitative RT-PCR, Isolation, Mouse Assay, Infection, In Vitro, RNA Binding Assay, Immunoprecipitation

    16) Product Images from "Erythritol Availability in Bovine, Murine and Human Models Highlights a Potential Role for the Host Aldose Reductase during Brucella Infection"

    Article Title: Erythritol Availability in Bovine, Murine and Human Models Highlights a Potential Role for the Host Aldose Reductase during Brucella Infection

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2017.01088

    The expression of Aldose reductase gene Akr1b3 in RAW 264.7 macrophages depends on glucose concentration. (A) Expression of gene Akr1b3 measured by qRTPCR in macrophages cultured with 0.5, 1, and 4.5 g/L of glucose. (B) Multiplication of B. abortus 2308 WT and Δ eryH in macrophages cultured with 0.5, 1, and 4.5 g/L of glucose. All experiments were performed in biological and technical duplicates ( ∗ p
    Figure Legend Snippet: The expression of Aldose reductase gene Akr1b3 in RAW 264.7 macrophages depends on glucose concentration. (A) Expression of gene Akr1b3 measured by qRTPCR in macrophages cultured with 0.5, 1, and 4.5 g/L of glucose. (B) Multiplication of B. abortus 2308 WT and Δ eryH in macrophages cultured with 0.5, 1, and 4.5 g/L of glucose. All experiments were performed in biological and technical duplicates ( ∗ p

    Techniques Used: Expressing, Concentration Assay, Cell Culture

    17) Product Images from "Blockade of vascular endothelial growth factor receptors by tivozanib has potential anti-tumour effects on human glioblastoma cells"

    Article Title: Blockade of vascular endothelial growth factor receptors by tivozanib has potential anti-tumour effects on human glioblastoma cells

    Journal: Scientific Reports

    doi: 10.1038/srep44075

    Tivozanib induces G2/M cell cycle arrest. ( A ) Following treatment with tivozanib for 48 h, the cell pellets were fixed and incubated with PI to analyse the cell cycle distribution on a flow cytometer. The graphs are representative of three independent experiments with similar results. ( B ) The cells were treated with tivozanib for 48 h then total RNA was harvested for qRT-PCR analysis. ( C ) Protein lysates from tivozanib-treated cells were subjected to Western blotting and probed with the indicated antibodies. β-actin was used as the loading control. The blots are representative of three independent experiments with similar outcomes. Data are given as mean ± SD. Statistically significant values of * p
    Figure Legend Snippet: Tivozanib induces G2/M cell cycle arrest. ( A ) Following treatment with tivozanib for 48 h, the cell pellets were fixed and incubated with PI to analyse the cell cycle distribution on a flow cytometer. The graphs are representative of three independent experiments with similar results. ( B ) The cells were treated with tivozanib for 48 h then total RNA was harvested for qRT-PCR analysis. ( C ) Protein lysates from tivozanib-treated cells were subjected to Western blotting and probed with the indicated antibodies. β-actin was used as the loading control. The blots are representative of three independent experiments with similar outcomes. Data are given as mean ± SD. Statistically significant values of * p

    Techniques Used: Incubation, Flow Cytometry, Cytometry, Quantitative RT-PCR, Western Blot

    Tivozanib inhibits adhesive and invasive potential of the GBM cells. ( A ) Tivozanib-treated cells were seeded into collagen I-coated culture dishes then the adhesive cells were stained, lysed and the optical densitometry was read. ( B,C ) The effects of tivozanib on mRNA and protein levels of ICAM-1 and VCAM-1 were measured by qRT-PCR and Western blot analysis. β-actin was used as the loading control. The blots are representative of three independent experiments with similar results. ( D ) Equal amounts of secreted protein from treated cells were incubated with a synthetic substrate labelled with amino-4-trifluoromethyl coumarin (AFC). The substrate is cleaved by cathepsin B to release AFC, which is fluorometrically detected. ( E ) The conditioned media from each sample was subjected to a chromogenic substrate, which is cleaved by active uPA and produces a colorimetrically detectable product. (F) The conditioned media was collected and separated on a non-reducing polyacrylamide gel containing gelatin A. Gelatinolytic activities are visualized as clear bands against the blue background of stained gelatin. The zymograms are representative of three independent experiments with similar results. The gels were cropped and the full-length gels are presented in Supplementary Fig. 3 . (G) The cells were placed into 8-μm porous culture inserts, treated with tivozanib and allowed to migrate for 48 h. The migrated cells on the lower surface of the inserts were fixed with methanol, stained with crystal violet, lysed with 30% acetic acid and the optical densitometry was measured at 590 nm. (H) For invasion assay, the cells were placed into matrigel-coated inserts and allowed to invade through the matrigel layer for 48 h. Data are given as mean ± SD. Statistically significant values of * p
    Figure Legend Snippet: Tivozanib inhibits adhesive and invasive potential of the GBM cells. ( A ) Tivozanib-treated cells were seeded into collagen I-coated culture dishes then the adhesive cells were stained, lysed and the optical densitometry was read. ( B,C ) The effects of tivozanib on mRNA and protein levels of ICAM-1 and VCAM-1 were measured by qRT-PCR and Western blot analysis. β-actin was used as the loading control. The blots are representative of three independent experiments with similar results. ( D ) Equal amounts of secreted protein from treated cells were incubated with a synthetic substrate labelled with amino-4-trifluoromethyl coumarin (AFC). The substrate is cleaved by cathepsin B to release AFC, which is fluorometrically detected. ( E ) The conditioned media from each sample was subjected to a chromogenic substrate, which is cleaved by active uPA and produces a colorimetrically detectable product. (F) The conditioned media was collected and separated on a non-reducing polyacrylamide gel containing gelatin A. Gelatinolytic activities are visualized as clear bands against the blue background of stained gelatin. The zymograms are representative of three independent experiments with similar results. The gels were cropped and the full-length gels are presented in Supplementary Fig. 3 . (G) The cells were placed into 8-μm porous culture inserts, treated with tivozanib and allowed to migrate for 48 h. The migrated cells on the lower surface of the inserts were fixed with methanol, stained with crystal violet, lysed with 30% acetic acid and the optical densitometry was measured at 590 nm. (H) For invasion assay, the cells were placed into matrigel-coated inserts and allowed to invade through the matrigel layer for 48 h. Data are given as mean ± SD. Statistically significant values of * p

    Techniques Used: Staining, Quantitative RT-PCR, Western Blot, Incubation, Invasion Assay

    18) Product Images from "Molecular Features and Expression Patterns of Vitellogenin Receptor in Calliptamus italicus (Orthoptera: Acrididae)"

    Article Title: Molecular Features and Expression Patterns of Vitellogenin Receptor in Calliptamus italicus (Orthoptera: Acrididae)

    Journal: Journal of Insect Science

    doi: 10.1093/jisesa/iez119

    Expression pattern of CiVgR in different developmental stages. The total RNA was extracted from the ovarian tissues of the fourth- and fifth-instar nymph of Calliptamus italicus (n4, n5), as well as 1- to 25-d-old female Calliptamus italicus after adult emergence (1–25); β-actin was used as the internal reference. The data were described with means ± standard divisions ( n = 3). The different letters indicate the statistically significant differences ( P
    Figure Legend Snippet: Expression pattern of CiVgR in different developmental stages. The total RNA was extracted from the ovarian tissues of the fourth- and fifth-instar nymph of Calliptamus italicus (n4, n5), as well as 1- to 25-d-old female Calliptamus italicus after adult emergence (1–25); β-actin was used as the internal reference. The data were described with means ± standard divisions ( n = 3). The different letters indicate the statistically significant differences ( P

    Techniques Used: Expressing

    19) Product Images from "One-step efficient generation of dual-function conditional knockout and geno-tagging alleles in zebrafish"

    Article Title: One-step efficient generation of dual-function conditional knockout and geno-tagging alleles in zebrafish

    Journal: eLife

    doi: 10.7554/eLife.48081

    Strategy and evaluation of the targeted insertion of the PoG-Ne donor at the kctd10 locus. ( A ) The position and sequence of the kctd10 intron 1 (I1) target site designed for the Cas9/gRNA system. The protospacer sequence is shown in red, and the PAM is shown in green. ( B ) Targeting efficiency evaluated by PCR and Hpy188I restriction endonuclease digestion. ( C ) Sequencing results of the uncut PCR products (corresponding to indel mutations) from B after cloning. ( D ) Schematic diagram of the kctd10-2A-td GFP floxP 2PA-mutExon PoNe donor (abbreviated as kctd10 PoG-Ne donor) and the strategy of targeted insertion and conditional knockout using the CRISPR/Cas system. Primers K10qF and K10qR are used for qRT-PCR in L and M. ( E ) Images of a 10 hpf F 0 zebrafish embryo after the injection of the kctd10 PoG-Ne donor together with zCas9 mRNA and corresponding gRNAs. White arrows indicate tdGFP signals. Scale bar, 200 μm. ( F ) Junction PCR to detect NHEJ-mediated knockin events in the injected founder embryos. Injected: Donor+Cas9/gRNA-injected embryos. Donor: kctd10 PoG-Ne donor plasmid. Uninjected: Uninjected embryos. ( G ) Images of a 10 hpf F 1 zebrafish embryo from an outcross of the kctd10 PoG-Ne donor KI-positive F 0 female (#32) shown in Supplementary file 4 , bearing the kctd10 PoG-Ne-1 allele. Strong maternal expression of tdGFP can be clearly observed in this F 1 embryo. Scale bar, 200 μm. ( H ) Schematic diagram of the kctd10 KI allele, showing the position of the primers used for junction PCR in I-K and qRT-PCR in L. A new primer pair was used to amplify the 3’ junction of the F 1 embryos. ( I ) Junction PCR to detect the knockin allele in individual F 1 embryos (1-4) from the cross in G. Note that not all of the embryos inherited the knockin allele from the F 0 female, indicating germline mosaicism of this adult fish. ( J ) Sequencing results of the PCR products from the two positive embryos (2 and 3) in I, which showed the same junction sequence of the kctd10 PoG-Ne-1 allele. ( K ) Sequencing results of the PCR products (using the same primer pair as in I and J) from an EGFP-positive F 1 zebrafish embryo obtained from an outcross of the positive F 0 male (#5), representing the junction sequence of the kctd10 PoG-Ne-2 allele. ( L ) qRT-PCR results showing the transcription level of the kctd10 locus in wild-type (WT) and kctd10 PoG-Ne donor KI zebrafish embryos at 72 hpf, using K10qF and K10qR primers. The kctd10 +/Ne-1 and kctd10 +/PoG-Ne-1 embryos were obtained from the cross of kctd10 PoG-Ne-1/PoG-Ne-1 homozygotes with wild-type zebrafish with or without the injection of Cre mRNA, respectively. The average expression level of wild-type embryos was set as 1. ( M ) qRT-PCR results using K10qF and K10qR primers, showing the transcription level of the kctd10 locus in the kctd10 +/Ne-1 and kctd10 Ne-1/Ne-1 embryos derived from the Cre mRNA-injected kctd10 +/PoG-Ne-1 and kctd10 PoG-Ne-1/PoG-Ne-1 embryos, respectively. The original embryos were obtained from the crossing of kctd10 PoG-Ne-1/PoG-Ne-1 homozygotes with kctd10 +/PoG-Ne-1 heterozygote zebrafish. The expression levels in the KI embryos were normalized to the WT ones. Data are presented as the mean ±s.d., and a two-tailed Student’s t -test was applied to calculate p values in all the experiments. *: p
    Figure Legend Snippet: Strategy and evaluation of the targeted insertion of the PoG-Ne donor at the kctd10 locus. ( A ) The position and sequence of the kctd10 intron 1 (I1) target site designed for the Cas9/gRNA system. The protospacer sequence is shown in red, and the PAM is shown in green. ( B ) Targeting efficiency evaluated by PCR and Hpy188I restriction endonuclease digestion. ( C ) Sequencing results of the uncut PCR products (corresponding to indel mutations) from B after cloning. ( D ) Schematic diagram of the kctd10-2A-td GFP floxP 2PA-mutExon PoNe donor (abbreviated as kctd10 PoG-Ne donor) and the strategy of targeted insertion and conditional knockout using the CRISPR/Cas system. Primers K10qF and K10qR are used for qRT-PCR in L and M. ( E ) Images of a 10 hpf F 0 zebrafish embryo after the injection of the kctd10 PoG-Ne donor together with zCas9 mRNA and corresponding gRNAs. White arrows indicate tdGFP signals. Scale bar, 200 μm. ( F ) Junction PCR to detect NHEJ-mediated knockin events in the injected founder embryos. Injected: Donor+Cas9/gRNA-injected embryos. Donor: kctd10 PoG-Ne donor plasmid. Uninjected: Uninjected embryos. ( G ) Images of a 10 hpf F 1 zebrafish embryo from an outcross of the kctd10 PoG-Ne donor KI-positive F 0 female (#32) shown in Supplementary file 4 , bearing the kctd10 PoG-Ne-1 allele. Strong maternal expression of tdGFP can be clearly observed in this F 1 embryo. Scale bar, 200 μm. ( H ) Schematic diagram of the kctd10 KI allele, showing the position of the primers used for junction PCR in I-K and qRT-PCR in L. A new primer pair was used to amplify the 3’ junction of the F 1 embryos. ( I ) Junction PCR to detect the knockin allele in individual F 1 embryos (1-4) from the cross in G. Note that not all of the embryos inherited the knockin allele from the F 0 female, indicating germline mosaicism of this adult fish. ( J ) Sequencing results of the PCR products from the two positive embryos (2 and 3) in I, which showed the same junction sequence of the kctd10 PoG-Ne-1 allele. ( K ) Sequencing results of the PCR products (using the same primer pair as in I and J) from an EGFP-positive F 1 zebrafish embryo obtained from an outcross of the positive F 0 male (#5), representing the junction sequence of the kctd10 PoG-Ne-2 allele. ( L ) qRT-PCR results showing the transcription level of the kctd10 locus in wild-type (WT) and kctd10 PoG-Ne donor KI zebrafish embryos at 72 hpf, using K10qF and K10qR primers. The kctd10 +/Ne-1 and kctd10 +/PoG-Ne-1 embryos were obtained from the cross of kctd10 PoG-Ne-1/PoG-Ne-1 homozygotes with wild-type zebrafish with or without the injection of Cre mRNA, respectively. The average expression level of wild-type embryos was set as 1. ( M ) qRT-PCR results using K10qF and K10qR primers, showing the transcription level of the kctd10 locus in the kctd10 +/Ne-1 and kctd10 Ne-1/Ne-1 embryos derived from the Cre mRNA-injected kctd10 +/PoG-Ne-1 and kctd10 PoG-Ne-1/PoG-Ne-1 embryos, respectively. The original embryos were obtained from the crossing of kctd10 PoG-Ne-1/PoG-Ne-1 homozygotes with kctd10 +/PoG-Ne-1 heterozygote zebrafish. The expression levels in the KI embryos were normalized to the WT ones. Data are presented as the mean ±s.d., and a two-tailed Student’s t -test was applied to calculate p values in all the experiments. *: p

    Techniques Used: Sequencing, Polymerase Chain Reaction, Clone Assay, Knock-Out, CRISPR, Quantitative RT-PCR, Injection, Non-Homologous End Joining, Knock-In, Plasmid Preparation, Expressing, Fluorescence In Situ Hybridization, Derivative Assay, Two Tailed Test

    Evaluation of the indel efficiency of the tbx5a E3 target site and phenotype analysis of the tbx5a indel mutation. ( A ) The position and sequence of the tbx5a exon 3 (E3) target site designed for the Cas9/gRNA system. The protospacer sequence is shown in red, and the PAM is shown in green. ( B ) Targeting efficiency evaluated by PCR and AluI restriction endonuclease digestion. The result indicates that the indel efficiency is nearly 90%. ( C ) Sequencing results of the uncut PCR products (corresponding to indel mutations) from B after cloning. ( D ) Approximately 25% of embryos from the incross of tbx5a +/Δ5 heterozygotes showed defects in heart (black arrows) and pectoral fins (black arrowheads). Genotyping results revealed that all the defective embryos were tbx5a Δ5/Δ5 homozygotes (lower panel), while the siblings showed a normal morphology. The Tg(cmlc2:EGFP) transgenic background was introduced to reveal the heart morphology, and all the defective embryos also showed failure of cardiac looping. The dotted lines denote the outline of the heart. Scale bar, 200 μm. ( E ) qRT-PCR results showing the transcription level of the tbx5a locus in wild-type (WT) and tbx5a PoR-Ne donor KI zebrafish embryos at 72 hpf, using T5qF and T5qR primers. The tbx5a +/Ne and tbx5a +/PoR-Ne embryos were obtained from the crossing of the tbx5a PoR-Ne/PoR-Ne homozygotes with wild-type zebrafish with or without injection of Cre mRNA, respectively. The average expression level of wild-type embryos was set as 1. ( F ) qRT-PCR results using T5qF and T5qR primers, showing the transcription level of the tbx5a locus in the tbx5a +/Ne and tbx5a Ne/Ne embryos derived from the Cre mRNA-injected tbx5a +/PoR-Ne and tbx5a PoR-Ne/PoR-Ne embryos, respectively. The original embryos were obtained from the crossing of tbx5a PoR-Ne/PoR-Ne homozygotes with tbx5a +/PoR-Ne heterozygote zebrafish. The expression levels in the KI embryos were normalized to the WT ones. Data are presented as the mean ±s.d., and a two-tailed Student’s t -test was applied to calculate p values in all the experiments. *: p
    Figure Legend Snippet: Evaluation of the indel efficiency of the tbx5a E3 target site and phenotype analysis of the tbx5a indel mutation. ( A ) The position and sequence of the tbx5a exon 3 (E3) target site designed for the Cas9/gRNA system. The protospacer sequence is shown in red, and the PAM is shown in green. ( B ) Targeting efficiency evaluated by PCR and AluI restriction endonuclease digestion. The result indicates that the indel efficiency is nearly 90%. ( C ) Sequencing results of the uncut PCR products (corresponding to indel mutations) from B after cloning. ( D ) Approximately 25% of embryos from the incross of tbx5a +/Δ5 heterozygotes showed defects in heart (black arrows) and pectoral fins (black arrowheads). Genotyping results revealed that all the defective embryos were tbx5a Δ5/Δ5 homozygotes (lower panel), while the siblings showed a normal morphology. The Tg(cmlc2:EGFP) transgenic background was introduced to reveal the heart morphology, and all the defective embryos also showed failure of cardiac looping. The dotted lines denote the outline of the heart. Scale bar, 200 μm. ( E ) qRT-PCR results showing the transcription level of the tbx5a locus in wild-type (WT) and tbx5a PoR-Ne donor KI zebrafish embryos at 72 hpf, using T5qF and T5qR primers. The tbx5a +/Ne and tbx5a +/PoR-Ne embryos were obtained from the crossing of the tbx5a PoR-Ne/PoR-Ne homozygotes with wild-type zebrafish with or without injection of Cre mRNA, respectively. The average expression level of wild-type embryos was set as 1. ( F ) qRT-PCR results using T5qF and T5qR primers, showing the transcription level of the tbx5a locus in the tbx5a +/Ne and tbx5a Ne/Ne embryos derived from the Cre mRNA-injected tbx5a +/PoR-Ne and tbx5a PoR-Ne/PoR-Ne embryos, respectively. The original embryos were obtained from the crossing of tbx5a PoR-Ne/PoR-Ne homozygotes with tbx5a +/PoR-Ne heterozygote zebrafish. The expression levels in the KI embryos were normalized to the WT ones. Data are presented as the mean ±s.d., and a two-tailed Student’s t -test was applied to calculate p values in all the experiments. *: p

    Techniques Used: Mutagenesis, Sequencing, Polymerase Chain Reaction, Clone Assay, Transgenic Assay, Quantitative RT-PCR, Injection, Expressing, Derivative Assay, Two Tailed Test

    Evaluation of the tbx5a geno-tagging effect. ( A ) Preselection of tbx5a geno-tagging F 0 individual by junction PCR analysis. 5’ or 3’ junctions were amplified by PCR using genomic DNA extracted from fin clips of the #1, #2, #9, #11, #24 and #42 F 0 adult fish. The corresponding primer pairs are shown on the left side of the gel images, and the positions of these primers can be found in Figure 3A . ( B ) Switching of fluorescent signals achieved from the tbx5a geno-tagging allele after Cre mRNA injection into the F 1 progeny from #42 positive F 0 outcrossed with a wild-type zebrafish. The arrowheads indicate pectoral fins. The outlined boxed areas indicate the heart region, showing the change in the fluorescent signals in the heart before and after Cre mRNA injection. Scale bar, 200 μm. ( C ) The experimental design for the functionality test of the tbx5a geno-tagging allele. The progeny from the cross of a tbx5a +/PoR-NeG heterozygote with a tbx5a +/PoR-Ne heterozygote were divided into three groups: Group I was injected with 100 pg Cre mRNA at the one-cell stage, Group II was injected with 25 pg Cre mRNA in a single cell at the 4 cell stage, and Group III remained untreated as a control. The histogram shows the ratio of defective embryos after Cre mRNA injection in different groups. ( D ) Confocal images of the heart regions of two embryos from the cross of Tg(cmlc2:zCreER T2 -2A-ECFP) transgenic fish with tbx5a PoR-NeG/PoR-NeG after 4-HT treatment, showing a red to green change in the fluorescent signals upon Cre induction. -S: Single-plane view, -M: Maximum intensity projection view of z-stack images. Scale bar, 50 μm. ( E ) qRT-PCR results showing the transcription level of the tbx5a locus in wild-type (WT) and tbx5a PoR-NeG geno-tagging donor KI zebrafish embryos at 72 hpf, using T5qF and T5qR primers. The tbx5a +/NeG and tbx5a +/PoR-NeG embryos were obtained from crosses of tbx5a PoR-NeG/PoR-NeG homozygotes with wild-type zebrafish with or without the injection of Cre mRNA, respectively. The average expression level of wild-type embryos was set as 1. ( F ) qRT-PCR results using T5qF and T5qR primers, showing the transcription level of the tbx5a locus in the tbx5a +/NeG and tbx5a NeG/NeG embryos derived from the Cre mRNA-injected tbx5a +/PoR-NeG and tbx5a PoR-NeG/PoR-NeG embryos, respectively. The original embryos were obtained from the crossing of tbx5a PoR-NeG/PoR-NeG homozygotes with tbx5a +/PoR-NeG heterozygote zebrafish. The expression levels in the KI embryos were normalized to the WT ones. Data are presented as the mean ±s.d., and a two-tailed Student’s t -test was applied to calculate p values in all the experiments. *: p
    Figure Legend Snippet: Evaluation of the tbx5a geno-tagging effect. ( A ) Preselection of tbx5a geno-tagging F 0 individual by junction PCR analysis. 5’ or 3’ junctions were amplified by PCR using genomic DNA extracted from fin clips of the #1, #2, #9, #11, #24 and #42 F 0 adult fish. The corresponding primer pairs are shown on the left side of the gel images, and the positions of these primers can be found in Figure 3A . ( B ) Switching of fluorescent signals achieved from the tbx5a geno-tagging allele after Cre mRNA injection into the F 1 progeny from #42 positive F 0 outcrossed with a wild-type zebrafish. The arrowheads indicate pectoral fins. The outlined boxed areas indicate the heart region, showing the change in the fluorescent signals in the heart before and after Cre mRNA injection. Scale bar, 200 μm. ( C ) The experimental design for the functionality test of the tbx5a geno-tagging allele. The progeny from the cross of a tbx5a +/PoR-NeG heterozygote with a tbx5a +/PoR-Ne heterozygote were divided into three groups: Group I was injected with 100 pg Cre mRNA at the one-cell stage, Group II was injected with 25 pg Cre mRNA in a single cell at the 4 cell stage, and Group III remained untreated as a control. The histogram shows the ratio of defective embryos after Cre mRNA injection in different groups. ( D ) Confocal images of the heart regions of two embryos from the cross of Tg(cmlc2:zCreER T2 -2A-ECFP) transgenic fish with tbx5a PoR-NeG/PoR-NeG after 4-HT treatment, showing a red to green change in the fluorescent signals upon Cre induction. -S: Single-plane view, -M: Maximum intensity projection view of z-stack images. Scale bar, 50 μm. ( E ) qRT-PCR results showing the transcription level of the tbx5a locus in wild-type (WT) and tbx5a PoR-NeG geno-tagging donor KI zebrafish embryos at 72 hpf, using T5qF and T5qR primers. The tbx5a +/NeG and tbx5a +/PoR-NeG embryos were obtained from crosses of tbx5a PoR-NeG/PoR-NeG homozygotes with wild-type zebrafish with or without the injection of Cre mRNA, respectively. The average expression level of wild-type embryos was set as 1. ( F ) qRT-PCR results using T5qF and T5qR primers, showing the transcription level of the tbx5a locus in the tbx5a +/NeG and tbx5a NeG/NeG embryos derived from the Cre mRNA-injected tbx5a +/PoR-NeG and tbx5a PoR-NeG/PoR-NeG embryos, respectively. The original embryos were obtained from the crossing of tbx5a PoR-NeG/PoR-NeG homozygotes with tbx5a +/PoR-NeG heterozygote zebrafish. The expression levels in the KI embryos were normalized to the WT ones. Data are presented as the mean ±s.d., and a two-tailed Student’s t -test was applied to calculate p values in all the experiments. *: p

    Techniques Used: Polymerase Chain Reaction, Amplification, Fluorescence In Situ Hybridization, Injection, Transgenic Assay, Quantitative RT-PCR, Expressing, Derivative Assay, Two Tailed Test

    Generation and evaluation of the sox10 geno-tagging allele. ( A ) The position and sequence of the sox10 intron 3 (I3) target site designed for the Cas9/gRNA system. The protospacer sequence is shown in red, and the PAM is shown in green. ( B ) Targeting efficiency evaluated by PCR and AciI restriction endonuclease digestion. The result indicates that the indel efficiency is nearly 85%. ( C ) Sequencing results of the uncut PCR products (corresponding to indel mutations) from B after cloning. ( D ) The donor design and geno-tagging KI strategy at the sox10 locus. Primers S10qF and S10qR are used for qRT-PCR in G and H. ( E ) Phenotype analysis of the 48 hpf F 2 embryos from the incrossing of sox10 +/PoR-NeG heterozygotes (derived from #6 F 0 ) after the injection of Cre mRNA at the one-cell stage. The upper panel shows an uninjected control embryo bearing red fluorescent signals with normal pigmentation, whose genotype should be either sox10 +/PoR-NeG or sox10 PoR-NeG/PoR-NeG . The middle panel represents one Cre -injected embryo showing slightly less pigmentation but with only green fluorescent signals, indicating an efficient switch to the expression of tdGFP from that of tdTomoto after Cre injection; therefore, the genotype should be sox10 +/NeG . The lower panel shows a Cre -injected embryo devoid of body pigmentation that faithfully recapitulates the expected phenotype of the sox10 loss-of-function mutation. Similar to the previous embryo, this embryo shows only green fluorescent signals due to the Cre -induced efficient switch of the expression of the fluorescent reporter gene; therefore, the genotype is most likely tbx5a NeG/NeG . The white arrowheads indicate otic vesicles, whose detailed structure can be seen under higher magnification of the boxed areas. Scale bar, 200 μm. ( F ) Genotyping results of the injected F 2 embryos in E determined via 5’ junction PCR analysis. Since all the defective embryos showed only green (tdGFP) and no red (tdTomoto) fluorescent signal, the PCR products are most likely derived from the amplification of the sox10 NeG allele. ( G ) qRT-PCR results showing the transcription level of the sox10 locus in wild-type (WT) and sox10 PoR-NeG geno-tagging donor KI zebrafish embryos at 72 hpf, using S10qF and S10qR primers. The sox10 +/NeG and sox10 +/PoR-NeG embryos were obtained from the crossing of sox10 PoR-NeG/PoR-NeG homozygotes with wild-type zebrafish with or without the injection of Cre mRNA, respectively. The average expression level of wild-type embryos was set as 1. ( H ) qRT-PCR results using S10qF and S10qR primers, showing the transcription level of the tbx5a locus in the sox10 +/NeG and sox10 NeG/NeG embryos derived from the Cre mRNA-injected sox10 +/PoR-NeG and sox10 PoR-NeG/PoR-NeG embryos, respectively. The original embryos were obtained from the crossing of sox10 PoR-NeG/PoR-NeG homozygotes with sox10 +/PoR-NeG heterozygote zebrafish. The expression levels in the KI embryos were normalized to the WT ones. Data are presented as the mean ±s.d., and a two-tailed Student’s t -test was applied to calculate p values in all the experiments. *: p
    Figure Legend Snippet: Generation and evaluation of the sox10 geno-tagging allele. ( A ) The position and sequence of the sox10 intron 3 (I3) target site designed for the Cas9/gRNA system. The protospacer sequence is shown in red, and the PAM is shown in green. ( B ) Targeting efficiency evaluated by PCR and AciI restriction endonuclease digestion. The result indicates that the indel efficiency is nearly 85%. ( C ) Sequencing results of the uncut PCR products (corresponding to indel mutations) from B after cloning. ( D ) The donor design and geno-tagging KI strategy at the sox10 locus. Primers S10qF and S10qR are used for qRT-PCR in G and H. ( E ) Phenotype analysis of the 48 hpf F 2 embryos from the incrossing of sox10 +/PoR-NeG heterozygotes (derived from #6 F 0 ) after the injection of Cre mRNA at the one-cell stage. The upper panel shows an uninjected control embryo bearing red fluorescent signals with normal pigmentation, whose genotype should be either sox10 +/PoR-NeG or sox10 PoR-NeG/PoR-NeG . The middle panel represents one Cre -injected embryo showing slightly less pigmentation but with only green fluorescent signals, indicating an efficient switch to the expression of tdGFP from that of tdTomoto after Cre injection; therefore, the genotype should be sox10 +/NeG . The lower panel shows a Cre -injected embryo devoid of body pigmentation that faithfully recapitulates the expected phenotype of the sox10 loss-of-function mutation. Similar to the previous embryo, this embryo shows only green fluorescent signals due to the Cre -induced efficient switch of the expression of the fluorescent reporter gene; therefore, the genotype is most likely tbx5a NeG/NeG . The white arrowheads indicate otic vesicles, whose detailed structure can be seen under higher magnification of the boxed areas. Scale bar, 200 μm. ( F ) Genotyping results of the injected F 2 embryos in E determined via 5’ junction PCR analysis. Since all the defective embryos showed only green (tdGFP) and no red (tdTomoto) fluorescent signal, the PCR products are most likely derived from the amplification of the sox10 NeG allele. ( G ) qRT-PCR results showing the transcription level of the sox10 locus in wild-type (WT) and sox10 PoR-NeG geno-tagging donor KI zebrafish embryos at 72 hpf, using S10qF and S10qR primers. The sox10 +/NeG and sox10 +/PoR-NeG embryos were obtained from the crossing of sox10 PoR-NeG/PoR-NeG homozygotes with wild-type zebrafish with or without the injection of Cre mRNA, respectively. The average expression level of wild-type embryos was set as 1. ( H ) qRT-PCR results using S10qF and S10qR primers, showing the transcription level of the tbx5a locus in the sox10 +/NeG and sox10 NeG/NeG embryos derived from the Cre mRNA-injected sox10 +/PoR-NeG and sox10 PoR-NeG/PoR-NeG embryos, respectively. The original embryos were obtained from the crossing of sox10 PoR-NeG/PoR-NeG homozygotes with sox10 +/PoR-NeG heterozygote zebrafish. The expression levels in the KI embryos were normalized to the WT ones. Data are presented as the mean ±s.d., and a two-tailed Student’s t -test was applied to calculate p values in all the experiments. *: p

    Techniques Used: Sequencing, Polymerase Chain Reaction, Clone Assay, Quantitative RT-PCR, Derivative Assay, Injection, Expressing, Mutagenesis, Amplification, Two Tailed Test

    20) Product Images from "The stringent stress response controls proteases and global regulators under optimal growth conditions in Pseudomonas aeruginosa"

    Article Title: The stringent stress response controls proteases and global regulators under optimal growth conditions in Pseudomonas aeruginosa

    Journal: bioRxiv

    doi: 10.1101/2020.05.23.112573

    Scatter plot of the correlation between RNA-Seq and qRT-PCR expression for genes around the threshold cut-off (FC ±1.5). Verification of RNA-Seq data was performed on the same RNA for the following 15 genes: PA0652, PA0675, PA0762, PA0893, PA1270, PA1282, PA1430, PA2237, PA2426, PA3385, PA3410, PA3462, PA3899, PA5261, and PA5344.
    Figure Legend Snippet: Scatter plot of the correlation between RNA-Seq and qRT-PCR expression for genes around the threshold cut-off (FC ±1.5). Verification of RNA-Seq data was performed on the same RNA for the following 15 genes: PA0652, PA0675, PA0762, PA0893, PA1270, PA1282, PA1430, PA2237, PA2426, PA3385, PA3410, PA3462, PA3899, PA5261, and PA5344.

    Techniques Used: RNA Sequencing Assay, Quantitative RT-PCR, Expressing

    21) Product Images from "MicroRNA-96 Promotes Schistosomiasis Hepatic Fibrosis in Mice by Suppressing Smad7"

    Article Title: MicroRNA-96 Promotes Schistosomiasis Hepatic Fibrosis in Mice by Suppressing Smad7

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1016/j.omtm.2018.10.002

    TGF-β1 Elevates miR-96 Expression through Post-transcriptional Regulation (A) qRT-PCR analysis of miR-96 expression in isolated hepatocytes, HSCs, and Kupffer cells from mouse livers at 0 and 50 dpi, and the miR-96 expression in HSCs of the wild-type mice at 0 and 50 dpi. (B) qRT-PCR analysis of tgf-β1 and il13 expression in the HSCs of infected mice. (C) qRT-PCR analysis of miR-96 and miR-214 expression in primary HSCs treated in vitro for 24 hr with IL10 (25 ng/mL), interferon (IFN)-γ (25 ng/mL), TGF-β1 (100 ng/mL), and IL13 (200 ng/mL). (D) qRT-PCR analysis of miR-96 expression in primary HSCs treated with different concentrations of TGF-β1 as indicated. (E) qRT-PCR analysis of miR-96 expression in primary HSCs treated with TGF-β1 (8 ng/mL) for various times. (F) qRT-PCR analysis of pri-miR-96 and pre-miR-96 expression levels in isolated HSCs from mice at 0 and 50 dpi. (G) qRT-PCR analysis of pri-miR-96 and pre-miR-96 expression in primary HSCs treated with TGF-β1 (10 ng/mL) for various times. (H) RNA-binding protein immunoprecipitation (RIP) analysis of post-transcriptional processing of miR-96 . The cell lysates of HSCs isolated from infected (50 dpi) and uninfected (0 dpi) mice were immunoprecipitated with anti-SMAD2+SMAD3, anti-DROSHA, or normal rabbit IgG and subjected to qRT-PCR analysis. (I) In vitro RIP analysis of post-transcriptional processing of miR-96 and miR-21 maturation. qRT-PCR analysis of miR-96 and miR-21 expression in primary HSCs treated with TGF-β1 (100 ng/mL) or IL13 (200 ng/mL) for 24 hr. HSC lysates were immunoprecipitated and subjected to qRT-PCR analysis as described above. Data are represented as mean ± SD. *p
    Figure Legend Snippet: TGF-β1 Elevates miR-96 Expression through Post-transcriptional Regulation (A) qRT-PCR analysis of miR-96 expression in isolated hepatocytes, HSCs, and Kupffer cells from mouse livers at 0 and 50 dpi, and the miR-96 expression in HSCs of the wild-type mice at 0 and 50 dpi. (B) qRT-PCR analysis of tgf-β1 and il13 expression in the HSCs of infected mice. (C) qRT-PCR analysis of miR-96 and miR-214 expression in primary HSCs treated in vitro for 24 hr with IL10 (25 ng/mL), interferon (IFN)-γ (25 ng/mL), TGF-β1 (100 ng/mL), and IL13 (200 ng/mL). (D) qRT-PCR analysis of miR-96 expression in primary HSCs treated with different concentrations of TGF-β1 as indicated. (E) qRT-PCR analysis of miR-96 expression in primary HSCs treated with TGF-β1 (8 ng/mL) for various times. (F) qRT-PCR analysis of pri-miR-96 and pre-miR-96 expression levels in isolated HSCs from mice at 0 and 50 dpi. (G) qRT-PCR analysis of pri-miR-96 and pre-miR-96 expression in primary HSCs treated with TGF-β1 (10 ng/mL) for various times. (H) RNA-binding protein immunoprecipitation (RIP) analysis of post-transcriptional processing of miR-96 . The cell lysates of HSCs isolated from infected (50 dpi) and uninfected (0 dpi) mice were immunoprecipitated with anti-SMAD2+SMAD3, anti-DROSHA, or normal rabbit IgG and subjected to qRT-PCR analysis. (I) In vitro RIP analysis of post-transcriptional processing of miR-96 and miR-21 maturation. qRT-PCR analysis of miR-96 and miR-21 expression in primary HSCs treated with TGF-β1 (100 ng/mL) or IL13 (200 ng/mL) for 24 hr. HSC lysates were immunoprecipitated and subjected to qRT-PCR analysis as described above. Data are represented as mean ± SD. *p

    Techniques Used: Expressing, Quantitative RT-PCR, Isolation, Mouse Assay, Infection, In Vitro, RNA Binding Assay, Immunoprecipitation

    22) Product Images from "Analyses of MicroRNA and mRNA Expression Profiles Reveal the Crucial Interaction Networks and Pathways for Regulation of Chicken Breast Muscle Development"

    Article Title: Analyses of MicroRNA and mRNA Expression Profiles Reveal the Crucial Interaction Networks and Pathways for Regulation of Chicken Breast Muscle Development

    Journal: Frontiers in Genetics

    doi: 10.3389/fgene.2019.00197

    Quantitative real-time PCR validation of differentially expressed miRNAs and their corresponding target mRNAs. The red dots indicate three biological repetition of gene in qRT-PCR result. The blue dots indicate three biological repetition of miRNA in qRT-PCR result. (A) The qRT-PCR validation of miR-30a-3p and its target gene FOXO3 ; (B) The qRT-PCR validation of miR-30a-3p and its target gene DYNLL2; (C) The qRT-PCR validation of miR-148a-3p and its target gene DYNLL2.
    Figure Legend Snippet: Quantitative real-time PCR validation of differentially expressed miRNAs and their corresponding target mRNAs. The red dots indicate three biological repetition of gene in qRT-PCR result. The blue dots indicate three biological repetition of miRNA in qRT-PCR result. (A) The qRT-PCR validation of miR-30a-3p and its target gene FOXO3 ; (B) The qRT-PCR validation of miR-30a-3p and its target gene DYNLL2; (C) The qRT-PCR validation of miR-148a-3p and its target gene DYNLL2.

    Techniques Used: Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    Quantitative real-time PCR (qRT-PCR) validation of differentially expressed mRNAs. The red dots indicate the biological repetition in qRT-PCR result. The blue dots indicate the biological repetition from RNA-seq.
    Figure Legend Snippet: Quantitative real-time PCR (qRT-PCR) validation of differentially expressed mRNAs. The red dots indicate the biological repetition in qRT-PCR result. The blue dots indicate the biological repetition from RNA-seq.

    Techniques Used: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, RNA Sequencing Assay

    23) Product Images from "Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering"

    Article Title: Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-33242-z

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal human Chondrocytes (hChondrocytes) and ( B ) hChondrocytes undergoing apoptosis or ( C ) both normal and apoptotic chondrocyte cell lines combined.
    Figure Legend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal human Chondrocytes (hChondrocytes) and ( B ) hChondrocytes undergoing apoptosis or ( C ) both normal and apoptotic chondrocyte cell lines combined.

    Techniques Used: Expressing, Quantitative RT-PCR

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm. ( A ) GeNorm results between Control cell groups with normal hChondrocytes, hBMSCs and hADSCs. ( B ) GeNorm results between treated cell groups, i.e. hChondrocytes undergoing apoptosis, chondrogenic differentiated hBMSCs and hADSCs. ( C ) GeNorm results between all untreated and treated cell lines and types.
    Figure Legend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm. ( A ) GeNorm results between Control cell groups with normal hChondrocytes, hBMSCs and hADSCs. ( B ) GeNorm results between treated cell groups, i.e. hChondrocytes undergoing apoptosis, chondrogenic differentiated hBMSCs and hADSCs. ( C ) GeNorm results between all untreated and treated cell lines and types.

    Techniques Used: Expressing, Quantitative RT-PCR

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hBMSCs and ( B ) chondrogenic differentiated hBMSCs separately or ( C ) both cell lines combined.
    Figure Legend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hBMSCs and ( B ) chondrogenic differentiated hBMSCs separately or ( C ) both cell lines combined.

    Techniques Used: Expressing, Quantitative RT-PCR

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) untreated rat rectus abdominis muscle tissue, ( B ) rat rectus abdominis muscle tissue treated with osteogenic medium, and ( C ) both normal and treated rat muscle tissue.
    Figure Legend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) untreated rat rectus abdominis muscle tissue, ( B ) rat rectus abdominis muscle tissue treated with osteogenic medium, and ( C ) both normal and treated rat muscle tissue.

    Techniques Used: Expressing, Quantitative RT-PCR

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hADSCs and ( B ) chondrogenic differentiated hADSCs separately or ( C ) both untreated and treated hADSCs cell lines combined.
    Figure Legend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hADSCs and ( B ) chondrogenic differentiated hADSCs separately or ( C ) both untreated and treated hADSCs cell lines combined.

    Techniques Used: Expressing, Quantitative RT-PCR

    24) Product Images from "Dacomitinib, a pan-inhibitor of ErbB receptors, suppresses growth and invasive capacity of chemoresistant ovarian carcinoma cells"

    Article Title: Dacomitinib, a pan-inhibitor of ErbB receptors, suppresses growth and invasive capacity of chemoresistant ovarian carcinoma cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-04147-0

    Dacomitinib inhibits PLK1-FOXM1 pathway. ( A ) HRG/HER3 loop activates PLK1. The effects of HRGβ-1 on PLK1 activation was determined by Western blot analysis. Protein lysates from serum-starved and HRGβ-1-treated cells were subjected to Western blotting and probed with the indicated antibodies. The blots are representative of three independent experiments with similar outcomes. ( B ) PLK1 blockade increases cisplatin sensitivity. The effects of BI 2536-cisplatin therapy on cell proliferation were investigated by MTT assay and shown by IC 50 shift analysis. The cultures were treated with BI 2536 (20 nM) and cisplatin (0.1, 0.5, 1, 2.5, 5 and 10 μg/mL) for 48 h. ( C ) Normalised isobolograms of combination of BI 2536 and cisplatin. ( D ) The effects of cetuximab (10 μg/mL), trastuzumab (10 μg/mL), H3.105.5 (10 μg/mL), erlotinib (5 μM) and dacomitinib (5 μM) on PLK1-FOXM1 pathway and its down-stream targets were determined by Western blot analysis. The blots are representative of three independent experiments with similar outcomes. ( E ) The effects of the anti-ErbB agents on the expression of PLK1-FOXM1 targets genes were determined by qRT-PCR analysis. Data shown represent the mean ± SD from three independent experiments, each performed in triplicate. Statistically significant values of * p
    Figure Legend Snippet: Dacomitinib inhibits PLK1-FOXM1 pathway. ( A ) HRG/HER3 loop activates PLK1. The effects of HRGβ-1 on PLK1 activation was determined by Western blot analysis. Protein lysates from serum-starved and HRGβ-1-treated cells were subjected to Western blotting and probed with the indicated antibodies. The blots are representative of three independent experiments with similar outcomes. ( B ) PLK1 blockade increases cisplatin sensitivity. The effects of BI 2536-cisplatin therapy on cell proliferation were investigated by MTT assay and shown by IC 50 shift analysis. The cultures were treated with BI 2536 (20 nM) and cisplatin (0.1, 0.5, 1, 2.5, 5 and 10 μg/mL) for 48 h. ( C ) Normalised isobolograms of combination of BI 2536 and cisplatin. ( D ) The effects of cetuximab (10 μg/mL), trastuzumab (10 μg/mL), H3.105.5 (10 μg/mL), erlotinib (5 μM) and dacomitinib (5 μM) on PLK1-FOXM1 pathway and its down-stream targets were determined by Western blot analysis. The blots are representative of three independent experiments with similar outcomes. ( E ) The effects of the anti-ErbB agents on the expression of PLK1-FOXM1 targets genes were determined by qRT-PCR analysis. Data shown represent the mean ± SD from three independent experiments, each performed in triplicate. Statistically significant values of * p

    Techniques Used: Activation Assay, Western Blot, MTT Assay, Expressing, Quantitative RT-PCR

    Dacomitinib inhibits migration and invasion. ( A ) The effects of the anti-ErbB agents on expression of EMT markers were determined by qRT-PCR analysis. ( B ) The effects of the ErbB inhibitors on cell migration and invasion. The cells were placed into 8-μm porous culture inserts, treated with the drugs and allowed to migrate for 48 h. The migrated cells on the lower surface of the inserts were quantified by crystal violet staining. For invasion assay, the cells were placed into matrigel-coated inserts and allowed to invade through the matrigel layer for 48 h. Data shown represent the mean ± SD from three independent experiments, each performed in triplicate. Statistically significant values of * p
    Figure Legend Snippet: Dacomitinib inhibits migration and invasion. ( A ) The effects of the anti-ErbB agents on expression of EMT markers were determined by qRT-PCR analysis. ( B ) The effects of the ErbB inhibitors on cell migration and invasion. The cells were placed into 8-μm porous culture inserts, treated with the drugs and allowed to migrate for 48 h. The migrated cells on the lower surface of the inserts were quantified by crystal violet staining. For invasion assay, the cells were placed into matrigel-coated inserts and allowed to invade through the matrigel layer for 48 h. Data shown represent the mean ± SD from three independent experiments, each performed in triplicate. Statistically significant values of * p

    Techniques Used: Migration, Expressing, Quantitative RT-PCR, Staining, Invasion Assay

    Expression of the ErbB family in the EOC cells. ( A , B ) The mRNA levels of the ErbB family in the EOC cell lines were determined by qRT-PCR. The data were evaluated in triplicate and collected from three independent experiments. Gene expression levels were normalised to B2M in each cell line. Data were analysed by one-way ANOVA followed by Tukey’s post hoc test and are shown as mean ± SD. Statistically significant values of * p
    Figure Legend Snippet: Expression of the ErbB family in the EOC cells. ( A , B ) The mRNA levels of the ErbB family in the EOC cell lines were determined by qRT-PCR. The data were evaluated in triplicate and collected from three independent experiments. Gene expression levels were normalised to B2M in each cell line. Data were analysed by one-way ANOVA followed by Tukey’s post hoc test and are shown as mean ± SD. Statistically significant values of * p

    Techniques Used: Expressing, Quantitative RT-PCR

    25) Product Images from "Direct and site-specific quantification of RNA 2′-O-methylation by PCR with an engineered DNA polymerase"

    Article Title: Direct and site-specific quantification of RNA 2′-O-methylation by PCR with an engineered DNA polymerase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw200

    DNA synthesis catalyzed by RT-KTQ-LSIM is hampered by 2′-O-methylation of RNA templates. ( A ) Structures of relevant nucleotides. ( B ) Primer extension in presence of methylated or unmethylated RNA templates catalyzed by RT-KTQ-LSIM. ( C ) qRT-PCR amplification of methylated and unmethylated RNA oligonucleotides catalyzed by RT-KTQ-LSIM.
    Figure Legend Snippet: DNA synthesis catalyzed by RT-KTQ-LSIM is hampered by 2′-O-methylation of RNA templates. ( A ) Structures of relevant nucleotides. ( B ) Primer extension in presence of methylated or unmethylated RNA templates catalyzed by RT-KTQ-LSIM. ( C ) qRT-PCR amplification of methylated and unmethylated RNA oligonucleotides catalyzed by RT-KTQ-LSIM.

    Techniques Used: DNA Synthesis, Methylation, Quantitative RT-PCR, Amplification

    RT-KTQ-LSIM V669L features increased discrimination between 2′-O-methylated and unmethlyated RNA templates and enables quantification of 2′-O-methylation by qRT-PCR. ( A ) Primer extension in the presence of methylated or unmethlyated RNA templates catalyzed by RT-KTQ-LSIM V669L. ( B ) qRT-PCR amplification of methylated and unmethylated RNA oligonucleotides catalyzed by RT-KTQ-LSIM V669L. ( C ) RT-PCR reactions were stopped after 25 cycles (top) or 30 cycles (bottom) and analyzed by agarose gel electrophoresis. ( D ) The ΔC T -method was used to calculate methylation ratio of RNA template at 100 pM concentration with varied fractions of 2′OmeA/A at the target position. Error bars describe SD (n = 3).
    Figure Legend Snippet: RT-KTQ-LSIM V669L features increased discrimination between 2′-O-methylated and unmethlyated RNA templates and enables quantification of 2′-O-methylation by qRT-PCR. ( A ) Primer extension in the presence of methylated or unmethlyated RNA templates catalyzed by RT-KTQ-LSIM V669L. ( B ) qRT-PCR amplification of methylated and unmethylated RNA oligonucleotides catalyzed by RT-KTQ-LSIM V669L. ( C ) RT-PCR reactions were stopped after 25 cycles (top) or 30 cycles (bottom) and analyzed by agarose gel electrophoresis. ( D ) The ΔC T -method was used to calculate methylation ratio of RNA template at 100 pM concentration with varied fractions of 2′OmeA/A at the target position. Error bars describe SD (n = 3).

    Techniques Used: Methylation, Quantitative RT-PCR, Amplification, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Concentration Assay

    Quantification of ribosomal methylation directly from total RNA by qRT-PCR. ( A ) Analysis of the methylation status of A27, A99, U428, G1490 and C1703 in 18s rRNA from total RNA extracts of various human cell lines. Error bars describe SD. ( B ) qRT-PCR data of methylation site A99 in HEK-293 and Caco2 cells. ΔC T values indicate higher degree of methylation in HEK-293 cells than in Caco2 cells.
    Figure Legend Snippet: Quantification of ribosomal methylation directly from total RNA by qRT-PCR. ( A ) Analysis of the methylation status of A27, A99, U428, G1490 and C1703 in 18s rRNA from total RNA extracts of various human cell lines. Error bars describe SD. ( B ) qRT-PCR data of methylation site A99 in HEK-293 and Caco2 cells. ΔC T values indicate higher degree of methylation in HEK-293 cells than in Caco2 cells.

    Techniques Used: Methylation, Quantitative RT-PCR

    26) Product Images from "ImprimatinC1, a novel plant immune-priming compound, functions as a partial agonist of salicylic acid"

    Article Title: ImprimatinC1, a novel plant immune-priming compound, functions as a partial agonist of salicylic acid

    Journal: Scientific Reports

    doi: 10.1038/srep00705

    Activity of imprimatinC1 as an SA analogue. (a) Activity regarding defence gene expression. The PR1 mRNA transcript levels were determined via qRT-PCR with cDNAs prepared from 10-day-old Arabidopsis seedlings (wild-type or sid2 ) soaked in liquid media containing 100 µM imprimatinC1 or SA for 24 hours. The expression values were normalised to Actin2 as an internal standard. These results are representative of three independent replicates. (b) Activity regarding feedback regulation of SA synthesis. ImprimatinC1 (100 µM) was incubated with Arabidopsis cells in suspension for 24 hours, and the cellular SA content was measured via LC-MS. The data are expressed as the mean ± SD (n = 3). (c) Activity regarding JA signalling. A solution of 100 µM imprimatinC1 or SA was applied to transgenic Arabidopsis seedlings harbouring the LOX2 promoter:: GUS gene construct along with 100 µM MeJA for 24 hours, and GUS activity was determined. (d) Schematic representation of the pleiotropic effects of SA and the point in the pathway at which imprimatinC1 acts.
    Figure Legend Snippet: Activity of imprimatinC1 as an SA analogue. (a) Activity regarding defence gene expression. The PR1 mRNA transcript levels were determined via qRT-PCR with cDNAs prepared from 10-day-old Arabidopsis seedlings (wild-type or sid2 ) soaked in liquid media containing 100 µM imprimatinC1 or SA for 24 hours. The expression values were normalised to Actin2 as an internal standard. These results are representative of three independent replicates. (b) Activity regarding feedback regulation of SA synthesis. ImprimatinC1 (100 µM) was incubated with Arabidopsis cells in suspension for 24 hours, and the cellular SA content was measured via LC-MS. The data are expressed as the mean ± SD (n = 3). (c) Activity regarding JA signalling. A solution of 100 µM imprimatinC1 or SA was applied to transgenic Arabidopsis seedlings harbouring the LOX2 promoter:: GUS gene construct along with 100 µM MeJA for 24 hours, and GUS activity was determined. (d) Schematic representation of the pleiotropic effects of SA and the point in the pathway at which imprimatinC1 acts.

    Techniques Used: Activity Assay, Expressing, Quantitative RT-PCR, Incubation, Liquid Chromatography with Mass Spectroscopy, Transgenic Assay, Construct

    Induction of defence genes by imprimatinC1 and its derivative molecules. The mRNA transcript levels of PR1 (a) , At2g14560 (b) and At2g41090 ( CaBP22 ) (c) were determined by qRT-PCR with cDNAs prepared from 10-day-old Arabidopsis seedlings soaked in liquid media containing 100 µM concentrations of the experimental compounds for 24 hours. The expression values were normalised to Actin2 as an internal standard. These results are representative of three independent replicates.
    Figure Legend Snippet: Induction of defence genes by imprimatinC1 and its derivative molecules. The mRNA transcript levels of PR1 (a) , At2g14560 (b) and At2g41090 ( CaBP22 ) (c) were determined by qRT-PCR with cDNAs prepared from 10-day-old Arabidopsis seedlings soaked in liquid media containing 100 µM concentrations of the experimental compounds for 24 hours. The expression values were normalised to Actin2 as an internal standard. These results are representative of three independent replicates.

    Techniques Used: Quantitative RT-PCR, Expressing

    Activity of imprimatinC1 as a partial agonist of SA. The mRNA transcript levels of PR1 (a) , At2g14560 (b) and At2g41090 ( CaBP22 ) (c) were determined by qRT-PCR with cDNAs prepared from 10-day-old Arabidopsis seedlings soaked in liquid media containing 100 µM concentrations of the experimental compounds for 24 hours. The expression values were normalised to Actin2 as an internal standard. These results are representative of three independent replicates.
    Figure Legend Snippet: Activity of imprimatinC1 as a partial agonist of SA. The mRNA transcript levels of PR1 (a) , At2g14560 (b) and At2g41090 ( CaBP22 ) (c) were determined by qRT-PCR with cDNAs prepared from 10-day-old Arabidopsis seedlings soaked in liquid media containing 100 µM concentrations of the experimental compounds for 24 hours. The expression values were normalised to Actin2 as an internal standard. These results are representative of three independent replicates.

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

    27) Product Images from "Direct and site-specific quantification of RNA 2′-O-methylation by PCR with an engineered DNA polymerase"

    Article Title: Direct and site-specific quantification of RNA 2′-O-methylation by PCR with an engineered DNA polymerase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw200

    Rational design of RT-KTQ-LSIM libraries. Amino acids in immediate proximity to the 2′-oxygen of the nucleotide paired to the incoming dNTP were selected for saturation mutagenesis (namely G668, V669, G672, R746, K747 and N750). Adapted from PDB 4BWM ( 24 ) using PyMOL (Schrödinger, LLC, New York, NY, USA).
    Figure Legend Snippet: Rational design of RT-KTQ-LSIM libraries. Amino acids in immediate proximity to the 2′-oxygen of the nucleotide paired to the incoming dNTP were selected for saturation mutagenesis (namely G668, V669, G672, R746, K747 and N750). Adapted from PDB 4BWM ( 24 ) using PyMOL (Schrödinger, LLC, New York, NY, USA).

    Techniques Used: Mutagenesis

    DNA synthesis catalyzed by RT-KTQ-LSIM is hampered by 2′-O-methylation of RNA templates. ( A ) Structures of relevant nucleotides. ( B ) Primer extension in presence of methylated or unmethylated RNA templates catalyzed by RT-KTQ-LSIM. ( C ) qRT-PCR amplification of methylated and unmethylated RNA oligonucleotides catalyzed by RT-KTQ-LSIM.
    Figure Legend Snippet: DNA synthesis catalyzed by RT-KTQ-LSIM is hampered by 2′-O-methylation of RNA templates. ( A ) Structures of relevant nucleotides. ( B ) Primer extension in presence of methylated or unmethylated RNA templates catalyzed by RT-KTQ-LSIM. ( C ) qRT-PCR amplification of methylated and unmethylated RNA oligonucleotides catalyzed by RT-KTQ-LSIM.

    Techniques Used: DNA Synthesis, Methylation, Quantitative RT-PCR, Amplification

    RT-KTQ-LSIM V669L features increased discrimination between 2′-O-methylated and unmethlyated RNA templates and enables quantification of 2′-O-methylation by qRT-PCR. ( A ) Primer extension in the presence of methylated or unmethlyated RNA templates catalyzed by RT-KTQ-LSIM V669L. ( B ) qRT-PCR amplification of methylated and unmethylated RNA oligonucleotides catalyzed by RT-KTQ-LSIM V669L. ( C ) RT-PCR reactions were stopped after 25 cycles (top) or 30 cycles (bottom) and analyzed by agarose gel electrophoresis. ( D ) The ΔC T -method was used to calculate methylation ratio of RNA template at 100 pM concentration with varied fractions of 2′OmeA/A at the target position. Error bars describe SD (n = 3).
    Figure Legend Snippet: RT-KTQ-LSIM V669L features increased discrimination between 2′-O-methylated and unmethlyated RNA templates and enables quantification of 2′-O-methylation by qRT-PCR. ( A ) Primer extension in the presence of methylated or unmethlyated RNA templates catalyzed by RT-KTQ-LSIM V669L. ( B ) qRT-PCR amplification of methylated and unmethylated RNA oligonucleotides catalyzed by RT-KTQ-LSIM V669L. ( C ) RT-PCR reactions were stopped after 25 cycles (top) or 30 cycles (bottom) and analyzed by agarose gel electrophoresis. ( D ) The ΔC T -method was used to calculate methylation ratio of RNA template at 100 pM concentration with varied fractions of 2′OmeA/A at the target position. Error bars describe SD (n = 3).

    Techniques Used: Methylation, Quantitative RT-PCR, Amplification, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Concentration Assay

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    Roche qrt pcr lightcycler 96 instrument
    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for <t>qRT-PCR</t> assays, utilizing the GeNorm algorithm, for ( A ) normal human Chondrocytes (hChondrocytes) and ( B ) hChondrocytes undergoing apoptosis or ( C ) both normal and apoptotic chondrocyte cell lines combined.
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    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal human Chondrocytes (hChondrocytes) and ( B ) hChondrocytes undergoing apoptosis or ( C ) both normal and apoptotic chondrocyte cell lines combined.

    Journal: Scientific Reports

    Article Title: Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering

    doi: 10.1038/s41598-018-33242-z

    Figure Lengend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal human Chondrocytes (hChondrocytes) and ( B ) hChondrocytes undergoing apoptosis or ( C ) both normal and apoptotic chondrocyte cell lines combined.

    Article Snippet: PCR reactions were carried out in 96-well plates in duplicate utilising a qRT-PCR LightCycler® 96 Instrument (Roche) with a total reaction volume of 10 μl, that contained 10 μM of each reference primer (Table ), 10 ng of cDNA and 2x FastStart Essential DNA Green Master (Roche).

    Techniques: Expressing, Quantitative RT-PCR

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm. ( A ) GeNorm results between Control cell groups with normal hChondrocytes, hBMSCs and hADSCs. ( B ) GeNorm results between treated cell groups, i.e. hChondrocytes undergoing apoptosis, chondrogenic differentiated hBMSCs and hADSCs. ( C ) GeNorm results between all untreated and treated cell lines and types.

    Journal: Scientific Reports

    Article Title: Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering

    doi: 10.1038/s41598-018-33242-z

    Figure Lengend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm. ( A ) GeNorm results between Control cell groups with normal hChondrocytes, hBMSCs and hADSCs. ( B ) GeNorm results between treated cell groups, i.e. hChondrocytes undergoing apoptosis, chondrogenic differentiated hBMSCs and hADSCs. ( C ) GeNorm results between all untreated and treated cell lines and types.

    Article Snippet: PCR reactions were carried out in 96-well plates in duplicate utilising a qRT-PCR LightCycler® 96 Instrument (Roche) with a total reaction volume of 10 μl, that contained 10 μM of each reference primer (Table ), 10 ng of cDNA and 2x FastStart Essential DNA Green Master (Roche).

    Techniques: Expressing, Quantitative RT-PCR

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hBMSCs and ( B ) chondrogenic differentiated hBMSCs separately or ( C ) both cell lines combined.

    Journal: Scientific Reports

    Article Title: Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering

    doi: 10.1038/s41598-018-33242-z

    Figure Lengend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hBMSCs and ( B ) chondrogenic differentiated hBMSCs separately or ( C ) both cell lines combined.

    Article Snippet: PCR reactions were carried out in 96-well plates in duplicate utilising a qRT-PCR LightCycler® 96 Instrument (Roche) with a total reaction volume of 10 μl, that contained 10 μM of each reference primer (Table ), 10 ng of cDNA and 2x FastStart Essential DNA Green Master (Roche).

    Techniques: Expressing, Quantitative RT-PCR

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) untreated rat rectus abdominis muscle tissue, ( B ) rat rectus abdominis muscle tissue treated with osteogenic medium, and ( C ) both normal and treated rat muscle tissue.

    Journal: Scientific Reports

    Article Title: Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering

    doi: 10.1038/s41598-018-33242-z

    Figure Lengend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) untreated rat rectus abdominis muscle tissue, ( B ) rat rectus abdominis muscle tissue treated with osteogenic medium, and ( C ) both normal and treated rat muscle tissue.

    Article Snippet: PCR reactions were carried out in 96-well plates in duplicate utilising a qRT-PCR LightCycler® 96 Instrument (Roche) with a total reaction volume of 10 μl, that contained 10 μM of each reference primer (Table ), 10 ng of cDNA and 2x FastStart Essential DNA Green Master (Roche).

    Techniques: Expressing, Quantitative RT-PCR

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hADSCs and ( B ) chondrogenic differentiated hADSCs separately or ( C ) both untreated and treated hADSCs cell lines combined.

    Journal: Scientific Reports

    Article Title: Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering

    doi: 10.1038/s41598-018-33242-z

    Figure Lengend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hADSCs and ( B ) chondrogenic differentiated hADSCs separately or ( C ) both untreated and treated hADSCs cell lines combined.

    Article Snippet: PCR reactions were carried out in 96-well plates in duplicate utilising a qRT-PCR LightCycler® 96 Instrument (Roche) with a total reaction volume of 10 μl, that contained 10 μM of each reference primer (Table ), 10 ng of cDNA and 2x FastStart Essential DNA Green Master (Roche).

    Techniques: Expressing, Quantitative RT-PCR

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm. ( A ) GeNorm results between Control cell groups with normal hChondrocytes, hBMSCs and hADSCs. ( B ) GeNorm results between treated cell groups, i.e. hChondrocytes undergoing apoptosis, chondrogenic differentiated hBMSCs and hADSCs. ( C ) GeNorm results between all untreated and treated cell lines and types.

    Journal: Scientific Reports

    Article Title: Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering

    doi: 10.1038/s41598-018-33242-z

    Figure Lengend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm. ( A ) GeNorm results between Control cell groups with normal hChondrocytes, hBMSCs and hADSCs. ( B ) GeNorm results between treated cell groups, i.e. hChondrocytes undergoing apoptosis, chondrogenic differentiated hBMSCs and hADSCs. ( C ) GeNorm results between all untreated and treated cell lines and types.

    Article Snippet: The PCR reactions were performed using a qRT-PCR LightCycler® 96 Instrument (Roche, Basel, Swiss), where the total volume per reaction was 10 μl, containing 10 μM of each reference primer (Table ), the corresponding diluted cDNA and 2x FastStart Essential DNA Green Master (Roche).

    Techniques: Expressing, Quantitative RT-PCR

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hADSCs and ( B ) chondrogenic differentiated hADSCs separately or ( C ) both untreated and treated hADSCs cell lines combined.

    Journal: Scientific Reports

    Article Title: Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering

    doi: 10.1038/s41598-018-33242-z

    Figure Lengend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hADSCs and ( B ) chondrogenic differentiated hADSCs separately or ( C ) both untreated and treated hADSCs cell lines combined.

    Article Snippet: The PCR reactions were performed using a qRT-PCR LightCycler® 96 Instrument (Roche, Basel, Swiss), where the total volume per reaction was 10 μl, containing 10 μM of each reference primer (Table ), the corresponding diluted cDNA and 2x FastStart Essential DNA Green Master (Roche).

    Techniques: Expressing, Quantitative RT-PCR

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal human Chondrocytes (hChondrocytes) and ( B ) hChondrocytes undergoing apoptosis or ( C ) both normal and apoptotic chondrocyte cell lines combined.

    Journal: Scientific Reports

    Article Title: Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering

    doi: 10.1038/s41598-018-33242-z

    Figure Lengend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal human Chondrocytes (hChondrocytes) and ( B ) hChondrocytes undergoing apoptosis or ( C ) both normal and apoptotic chondrocyte cell lines combined.

    Article Snippet: The PCR reactions were performed using a qRT-PCR LightCycler® 96 Instrument (Roche, Basel, Swiss), where the total volume per reaction was 10 μl, containing 10 μM of each reference primer (Table ), the corresponding diluted cDNA and 2x FastStart Essential DNA Green Master (Roche).

    Techniques: Expressing, Quantitative RT-PCR

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hBMSCs and ( B ) chondrogenic differentiated hBMSCs separately or ( C ) both cell lines combined.

    Journal: Scientific Reports

    Article Title: Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering

    doi: 10.1038/s41598-018-33242-z

    Figure Lengend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hBMSCs and ( B ) chondrogenic differentiated hBMSCs separately or ( C ) both cell lines combined.

    Article Snippet: The PCR reactions were performed using a qRT-PCR LightCycler® 96 Instrument (Roche, Basel, Swiss), where the total volume per reaction was 10 μl, containing 10 μM of each reference primer (Table ), the corresponding diluted cDNA and 2x FastStart Essential DNA Green Master (Roche).

    Techniques: Expressing, Quantitative RT-PCR

    Quantitative real-time PCR validation of differentially expressed miRNAs and their corresponding target mRNAs. The red dots indicate three biological repetition of gene in qRT-PCR result. The blue dots indicate three biological repetition of miRNA in qRT-PCR result. (A) The qRT-PCR validation of miR-30a-3p and its target gene FOXO3 ; (B) The qRT-PCR validation of miR-30a-3p and its target gene DYNLL2; (C) The qRT-PCR validation of miR-148a-3p and its target gene DYNLL2.

    Journal: Frontiers in Genetics

    Article Title: Analyses of MicroRNA and mRNA Expression Profiles Reveal the Crucial Interaction Networks and Pathways for Regulation of Chicken Breast Muscle Development

    doi: 10.3389/fgene.2019.00197

    Figure Lengend Snippet: Quantitative real-time PCR validation of differentially expressed miRNAs and their corresponding target mRNAs. The red dots indicate three biological repetition of gene in qRT-PCR result. The blue dots indicate three biological repetition of miRNA in qRT-PCR result. (A) The qRT-PCR validation of miR-30a-3p and its target gene FOXO3 ; (B) The qRT-PCR validation of miR-30a-3p and its target gene DYNLL2; (C) The qRT-PCR validation of miR-148a-3p and its target gene DYNLL2.

    Article Snippet: Quantitative Real-Time PCR (qRT-PCR) Analysis For the qRT-PCR analysis of genes, reverse transcription was performed using a PrimerScriptTM RT reagent Kit (TaKaRa, Dalian, China) according to the manufacturer’s instructions. qRT-PCR with iTaqTM Universal SYBR® Green Supermix Kit (Bio-Rad Laboratories Inc., Waltham, MA, United States) was performed with a LightCycler® 96 instrument qRT-PCR system (Roche, Basel, Switzerland) as follows: 95°C for 3 min; 40 cycles of 95°C for 10 s, annealing at 60°C for 30 s, and 72°C for 30 s, and 72°C for 1 min.

    Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    Quantitative real-time PCR (qRT-PCR) validation of differentially expressed mRNAs. The red dots indicate the biological repetition in qRT-PCR result. The blue dots indicate the biological repetition from RNA-seq.

    Journal: Frontiers in Genetics

    Article Title: Analyses of MicroRNA and mRNA Expression Profiles Reveal the Crucial Interaction Networks and Pathways for Regulation of Chicken Breast Muscle Development

    doi: 10.3389/fgene.2019.00197

    Figure Lengend Snippet: Quantitative real-time PCR (qRT-PCR) validation of differentially expressed mRNAs. The red dots indicate the biological repetition in qRT-PCR result. The blue dots indicate the biological repetition from RNA-seq.

    Article Snippet: Quantitative Real-Time PCR (qRT-PCR) Analysis For the qRT-PCR analysis of genes, reverse transcription was performed using a PrimerScriptTM RT reagent Kit (TaKaRa, Dalian, China) according to the manufacturer’s instructions. qRT-PCR with iTaqTM Universal SYBR® Green Supermix Kit (Bio-Rad Laboratories Inc., Waltham, MA, United States) was performed with a LightCycler® 96 instrument qRT-PCR system (Roche, Basel, Switzerland) as follows: 95°C for 3 min; 40 cycles of 95°C for 10 s, annealing at 60°C for 30 s, and 72°C for 30 s, and 72°C for 1 min.

    Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, RNA Sequencing Assay