tamoxifen  (Millipore)


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    Name:
    Tamoxifen
    Description:
    Tamoxifen is a selective estrogen response modifier SERM protein kinase C inhibitor and anti angiogenetic factor Tamoxifen is a prodrug that is metabolized to active metabolites 4 hydroxytamoxifen 4 OHT and endoxifen by cytochrome P450 isoforms CYP2D6 and CYP3A4 In breast cancer the gene repressor activity of tamoxifen against ERBB2 is dependent upon PAX2 Blocks estradiol stimulated VEGF production in breast tumor cells
    Catalog Number:
    t5648
    Price:
    None
    Applications:
    Tamoxifen has been used to facilitate the recombination of ect2flox allele in mouse organs91. It has also been used to study its effect on lipopolysaccharide (LPS)-induced microglial activation92.
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    Structured Review

    Millipore tamoxifen
    Tamoxifen
    Tamoxifen is a selective estrogen response modifier SERM protein kinase C inhibitor and anti angiogenetic factor Tamoxifen is a prodrug that is metabolized to active metabolites 4 hydroxytamoxifen 4 OHT and endoxifen by cytochrome P450 isoforms CYP2D6 and CYP3A4 In breast cancer the gene repressor activity of tamoxifen against ERBB2 is dependent upon PAX2 Blocks estradiol stimulated VEGF production in breast tumor cells
    https://www.bioz.com/result/tamoxifen/product/Millipore
    Average 99 stars, based on 386 article reviews
    Price from $9.99 to $1999.99
    tamoxifen - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Co-activation of NF-κB and MYC renders cancer cells addicted to IL6 for survival and phenotypic stability"

    Article Title: Co-activation of NF-κB and MYC renders cancer cells addicted to IL6 for survival and phenotypic stability

    Journal: bioRxiv

    doi: 10.1101/2020.04.12.038414

    Dynamics of reporter positive subpopulations over-time. (A) Representative flow cytometric analysis of Cre-mediated recombination in the blood of C-YFP, C-MYC, C-IKK2 , and C-IM at days 55, 95, 135, 175 after the first tamoxifen administration. (B) Fraction of cells within the individual reporter positive populations: YFP + in C-YFP mice and GFP neg hCD2 + i.e. MYC + , GFP + hCD2 neg i.e. IKK2ca + , and GFP + hCD2 + i.e. IKK2ca + MYC + in C-IM mice. Frequencies were normalized to day 55 after the first tamoxifen administration. (C) Left, fraction of GFP neg hCD2 + i.e. MYC + in C-MYC mice. Right, fraction of GFP + hCD2 neg i.e. IKK2ca + in C-IKK2 mice. Frequencies were normalized to day 55 after the first tamoxifen administration.
    Figure Legend Snippet: Dynamics of reporter positive subpopulations over-time. (A) Representative flow cytometric analysis of Cre-mediated recombination in the blood of C-YFP, C-MYC, C-IKK2 , and C-IM at days 55, 95, 135, 175 after the first tamoxifen administration. (B) Fraction of cells within the individual reporter positive populations: YFP + in C-YFP mice and GFP neg hCD2 + i.e. MYC + , GFP + hCD2 neg i.e. IKK2ca + , and GFP + hCD2 + i.e. IKK2ca + MYC + in C-IM mice. Frequencies were normalized to day 55 after the first tamoxifen administration. (C) Left, fraction of GFP neg hCD2 + i.e. MYC + in C-MYC mice. Right, fraction of GFP + hCD2 neg i.e. IKK2ca + in C-IKK2 mice. Frequencies were normalized to day 55 after the first tamoxifen administration.

    Techniques Used: Mouse Assay

    Analysis of C-IM-IL6KO mice. (A) Left, frequency in bone marrow of C-IM and C-IM-IL6KO mice at day 100 after first tamoxifen injection of reporter positive populations: GFP neg hCD2 + i.e. MYC + , GFP + hCD2 neg i.e. IKK2ca + , and GFP + hCD2 + i.e. IKK2ca + MYC + . Right, frequency in bone marrow of C-IM and C-IM-IL6KO mice at day 100 after first tamoxifen injection of CD19 low CD138 + cells within each reporter positive populations and within reporter negative cells. (B) Representative histological and immunohistochemical analysis of a B-cell cancer in the spleen of a C-IM-IL6KO mice (top panels) and of a Plasma-cell cancer in the spleen of C-IM-IL6KO mice (bottom panels) for H E, B220, Pax5, Irf4, and Ki67.
    Figure Legend Snippet: Analysis of C-IM-IL6KO mice. (A) Left, frequency in bone marrow of C-IM and C-IM-IL6KO mice at day 100 after first tamoxifen injection of reporter positive populations: GFP neg hCD2 + i.e. MYC + , GFP + hCD2 neg i.e. IKK2ca + , and GFP + hCD2 + i.e. IKK2ca + MYC + . Right, frequency in bone marrow of C-IM and C-IM-IL6KO mice at day 100 after first tamoxifen injection of CD19 low CD138 + cells within each reporter positive populations and within reporter negative cells. (B) Representative histological and immunohistochemical analysis of a B-cell cancer in the spleen of a C-IM-IL6KO mice (top panels) and of a Plasma-cell cancer in the spleen of C-IM-IL6KO mice (bottom panels) for H E, B220, Pax5, Irf4, and Ki67.

    Techniques Used: Mouse Assay, Injection, Immunohistochemistry

    NF-κB signaling and MYC over-expression synergize for hyperplasia of a Plasma-cell like population. See also Fig S1. (A-B) Scheme illustrating the genetic systems used in the study. Triangles represent loxP sites. R26p and CAGp represent Rosa26 promoter and CAG promoter. (B) Schematic representation of the protocol of the study. Black arrow: immunization time-point with sheep red blood cells (SRBC); red arrow: day counting after the first tamoxifen administration; Blue arrow: tamoxifen administration time-point; yellow arrow: analysis time-point. (D-F) Representative flow cytometric analysis of Cre-mediated recombination in C-YFP and C-IM mice at day 10 (D), day 55 (E) and day 100 (F) after the first tamoxifen administration. Histograms represent CD19 expression within the recombined populations and bottom panels illustrate B-cell (CD19 + ) and Plasma-cell like (CD19 low CD138 + ) populations within the individual reporter positive populations: GFP neg hCD2 + i.e. MYC + , GFP + hCD2 neg i.e. IKK2ca + , and GFP + hCD2 + i.e. IKK2ca + MYC + .
    Figure Legend Snippet: NF-κB signaling and MYC over-expression synergize for hyperplasia of a Plasma-cell like population. See also Fig S1. (A-B) Scheme illustrating the genetic systems used in the study. Triangles represent loxP sites. R26p and CAGp represent Rosa26 promoter and CAG promoter. (B) Schematic representation of the protocol of the study. Black arrow: immunization time-point with sheep red blood cells (SRBC); red arrow: day counting after the first tamoxifen administration; Blue arrow: tamoxifen administration time-point; yellow arrow: analysis time-point. (D-F) Representative flow cytometric analysis of Cre-mediated recombination in C-YFP and C-IM mice at day 10 (D), day 55 (E) and day 100 (F) after the first tamoxifen administration. Histograms represent CD19 expression within the recombined populations and bottom panels illustrate B-cell (CD19 + ) and Plasma-cell like (CD19 low CD138 + ) populations within the individual reporter positive populations: GFP neg hCD2 + i.e. MYC + , GFP + hCD2 neg i.e. IKK2ca + , and GFP + hCD2 + i.e. IKK2ca + MYC + .

    Techniques Used: Over Expression, Mouse Assay, Expressing

    IL6 is critical for the formation of NF-κB + MYC + cancers at a poorly-differentiated Plasma-cell state. See also Fig S4. (A) Representative flow cytometric analysis of Cre-mediated recombination in in spleen of C-IM and C-IM-IL6KO mice at day 100 after the first tamoxifen administration (protocol of study as in Fig. 1C ). (B) Frequency in spleen of reporter positive populations: GFP neg hCD2 + i.e. MYC + , GFP + hCD2 neg i.e. IKK2ca + , and GFP + hCD2 + i.e. IKK2ca + MYC + . (C) Frequency in spleen of CD19 low CD138 + cells within each reporter positive populations as in (B) and within reporter negative cells. (D) Frequency in spleen of cleaved caspase 3 + CD19 low CD138 + cells within each reporter positive populations as in (B) and within reporter negative cells. (E) Frequency in spleen of CD19 low CD138 + cells at the S/G2M phase of the cell cycle within each reporter positive populations as in (B) and within reporter negative cells. (F) Cancer free survival curve for C-IM and C-IM-IL6KO mice. (G) Frequency of cleaved caspase-3 + within CD19 low CD138 + cancer cells of spleen of C-IM and C-IM-IL6KO . (H) Enrichment for genes in the signature “Hallmark_Apoptosis” using GSEA in the GEP of C-IM-IL6KO and C-IM cancer cells (GFP + hCD2 + FACS sorted). (I) Transcriptional analysis of C-IM and C-IM-IL6KO cancer cells compared to discrete B-cell and Plasma-cell populations by RNAseq. GCB: Germinal Center B-cells, B1: B1 B-cells, FOB: Follicular B-cells, MZB: Marginal Zone B-cells, SPLPC: Spleen Plasma-cells, BMPC: Bone Marrow Plasma-cells, SPLPB: Spleen Plasmablasts ( Shi et al., 2015 ). Seven C-IM and six C-IM-IL6KO cancers are depicted. B-cell signature: expression profile of the top 50 downregulated genes in BMPCs compared with FoBs, Plasma-cell signature: expression profile of the top 50 upregulated genes in BMPCs compared to FOBs, in addition to 4 genes of particular immunological interest ( Slc3a2, Prdm1, Ly6c1, Cd28 ). Log2 FPKM expression values of genes are shown in the heatmaps, color-coded according to the legend. (J) Analysis of clonality by RNA sequencing. The fraction of reads mapped to each individual IgH-V gene out of all the reads mapped to IgH-V genes is shown ( Shi et al., 2015 ). Top panel: a GFP + hCD2 + C-IM-IL6KO B-cell cancer, bottom panel: a GFP + hCD2 + C-IM-IL6KO Plasma-cell cancer. (K) Enrichment for genes in the signature “Bcell_signature_Shi” using GSEA of the GEP of C-IM and C-IM-IL6KO (B-cell cancers) cancer cells. (L) RNAseq expression data for factors related to Plasma-cell differentiation ( Prdm1, Irf4, Xpb1 ) and B-cell identity ( Pax5 ) in cancer cells of C-IM and C-IM-IL6KO (B-cell cancers). (M) Enrichment for genes in the signature “Plasmablast_signature_Shi” using GSEA of the GEP of C-IM and C-IM-IL6KO (Plasma-cell cancers) cancer cells. (N) RNAseq expression data for factors related to Plasma-cell differentiation ( Prdm1, Irf4, Xpb1 ) and B-cell identity ( Pax5 ) in cancer cells of C-IM and C-IM-IL6KO (Plasma-cell cancers).
    Figure Legend Snippet: IL6 is critical for the formation of NF-κB + MYC + cancers at a poorly-differentiated Plasma-cell state. See also Fig S4. (A) Representative flow cytometric analysis of Cre-mediated recombination in in spleen of C-IM and C-IM-IL6KO mice at day 100 after the first tamoxifen administration (protocol of study as in Fig. 1C ). (B) Frequency in spleen of reporter positive populations: GFP neg hCD2 + i.e. MYC + , GFP + hCD2 neg i.e. IKK2ca + , and GFP + hCD2 + i.e. IKK2ca + MYC + . (C) Frequency in spleen of CD19 low CD138 + cells within each reporter positive populations as in (B) and within reporter negative cells. (D) Frequency in spleen of cleaved caspase 3 + CD19 low CD138 + cells within each reporter positive populations as in (B) and within reporter negative cells. (E) Frequency in spleen of CD19 low CD138 + cells at the S/G2M phase of the cell cycle within each reporter positive populations as in (B) and within reporter negative cells. (F) Cancer free survival curve for C-IM and C-IM-IL6KO mice. (G) Frequency of cleaved caspase-3 + within CD19 low CD138 + cancer cells of spleen of C-IM and C-IM-IL6KO . (H) Enrichment for genes in the signature “Hallmark_Apoptosis” using GSEA in the GEP of C-IM-IL6KO and C-IM cancer cells (GFP + hCD2 + FACS sorted). (I) Transcriptional analysis of C-IM and C-IM-IL6KO cancer cells compared to discrete B-cell and Plasma-cell populations by RNAseq. GCB: Germinal Center B-cells, B1: B1 B-cells, FOB: Follicular B-cells, MZB: Marginal Zone B-cells, SPLPC: Spleen Plasma-cells, BMPC: Bone Marrow Plasma-cells, SPLPB: Spleen Plasmablasts ( Shi et al., 2015 ). Seven C-IM and six C-IM-IL6KO cancers are depicted. B-cell signature: expression profile of the top 50 downregulated genes in BMPCs compared with FoBs, Plasma-cell signature: expression profile of the top 50 upregulated genes in BMPCs compared to FOBs, in addition to 4 genes of particular immunological interest ( Slc3a2, Prdm1, Ly6c1, Cd28 ). Log2 FPKM expression values of genes are shown in the heatmaps, color-coded according to the legend. (J) Analysis of clonality by RNA sequencing. The fraction of reads mapped to each individual IgH-V gene out of all the reads mapped to IgH-V genes is shown ( Shi et al., 2015 ). Top panel: a GFP + hCD2 + C-IM-IL6KO B-cell cancer, bottom panel: a GFP + hCD2 + C-IM-IL6KO Plasma-cell cancer. (K) Enrichment for genes in the signature “Bcell_signature_Shi” using GSEA of the GEP of C-IM and C-IM-IL6KO (B-cell cancers) cancer cells. (L) RNAseq expression data for factors related to Plasma-cell differentiation ( Prdm1, Irf4, Xpb1 ) and B-cell identity ( Pax5 ) in cancer cells of C-IM and C-IM-IL6KO (B-cell cancers). (M) Enrichment for genes in the signature “Plasmablast_signature_Shi” using GSEA of the GEP of C-IM and C-IM-IL6KO (Plasma-cell cancers) cancer cells. (N) RNAseq expression data for factors related to Plasma-cell differentiation ( Prdm1, Irf4, Xpb1 ) and B-cell identity ( Pax5 ) in cancer cells of C-IM and C-IM-IL6KO (Plasma-cell cancers).

    Techniques Used: Mouse Assay, FACS, Expressing, RNA Sequencing Assay, Cell Differentiation

    2) Product Images from "Control of microglial dynamics by Arp2/3 and the autism and schizophrenia-associated protein Cyfip1"

    Article Title: Control of microglial dynamics by Arp2/3 and the autism and schizophrenia-associated protein Cyfip1

    Journal: bioRxiv

    doi: 10.1101/2020.05.31.124941

    Generation of a microglia-specific Cyfip1 conditional knockout mouse model (A) mRNA expression of WRC components, showing log(fold change) (logFC) of FPKM values from microglia and whole brain samples. Positive logFC values indicates enrichment in microglia. Adapted from Zhang et al. (2014) . See also Fig.S3 (B) Schematic showing generation of conditional Cyfip1 deletion in microglia using the Cre ERT2 driven from the microglia-specific CX3CR1 promoter. Cre was activated through treatment of experimental mice with tamoxifen. See also Fig.S4 (C) PCR confirmation of recombination of the floxed Cyfip1 cassette in control and Cyfip1 cKO mice. (D) Western blot for Cyfip1 in an enriched microglial extract from adult mouse brain, quantification in the right-hand panel (control: 1 ± 0.15, cKO: 0.48 ± 0.06; p
    Figure Legend Snippet: Generation of a microglia-specific Cyfip1 conditional knockout mouse model (A) mRNA expression of WRC components, showing log(fold change) (logFC) of FPKM values from microglia and whole brain samples. Positive logFC values indicates enrichment in microglia. Adapted from Zhang et al. (2014) . See also Fig.S3 (B) Schematic showing generation of conditional Cyfip1 deletion in microglia using the Cre ERT2 driven from the microglia-specific CX3CR1 promoter. Cre was activated through treatment of experimental mice with tamoxifen. See also Fig.S4 (C) PCR confirmation of recombination of the floxed Cyfip1 cassette in control and Cyfip1 cKO mice. (D) Western blot for Cyfip1 in an enriched microglial extract from adult mouse brain, quantification in the right-hand panel (control: 1 ± 0.15, cKO: 0.48 ± 0.06; p

    Techniques Used: Knock-Out, Expressing, Mouse Assay, Polymerase Chain Reaction, Western Blot

    Generation of Cyfip1 conditional knockout mouse model (A) Schematic illustrating the genetic modifications used to generate the Cyfip1 conditional knockout (cKO) mouse model. A floxed Cyfip1 line was generated using a knockout-first strategy and bred to homozygosity in all parental mice. To generate knockout alleles, floxed Cyfip1 mice were is crossed a knock-in CX3Cr1 CreERT2 line. CX3Cr1 CreERT2 allele was kept heterozygous in all breeding and experimental animals. An IBA GFP line used previously was crossed in to fluorescently label microglia. Recombination of the floxed Cyfip1 locus excises critical exons 4-6, leading to loss of functional Cyfip1 . Nuclear translocation of Cre recombinase was activated by tamoxifen treatment and was inactive in vehicle treatment. Small arrowheads identify approximate sites for PCR primer sequences for wildtype (Wt F /Wt R ), conditional (Wt F /Mut R ) and the deletion (Del F /Del R ) alleles of the Cyfip1 locus. Grey arrows denote no PCR product.
    Figure Legend Snippet: Generation of Cyfip1 conditional knockout mouse model (A) Schematic illustrating the genetic modifications used to generate the Cyfip1 conditional knockout (cKO) mouse model. A floxed Cyfip1 line was generated using a knockout-first strategy and bred to homozygosity in all parental mice. To generate knockout alleles, floxed Cyfip1 mice were is crossed a knock-in CX3Cr1 CreERT2 line. CX3Cr1 CreERT2 allele was kept heterozygous in all breeding and experimental animals. An IBA GFP line used previously was crossed in to fluorescently label microglia. Recombination of the floxed Cyfip1 locus excises critical exons 4-6, leading to loss of functional Cyfip1 . Nuclear translocation of Cre recombinase was activated by tamoxifen treatment and was inactive in vehicle treatment. Small arrowheads identify approximate sites for PCR primer sequences for wildtype (Wt F /Wt R ), conditional (Wt F /Mut R ) and the deletion (Del F /Del R ) alleles of the Cyfip1 locus. Grey arrows denote no PCR product.

    Techniques Used: Knock-Out, Generated, Mouse Assay, Knock-In, Functional Assay, Translocation Assay, Polymerase Chain Reaction

    3) Product Images from "Sonic hedgehog signaling in astrocytes mediates cell type-specific synaptic organization"

    Article Title: Sonic hedgehog signaling in astrocytes mediates cell type-specific synaptic organization

    Journal: bioRxiv

    doi: 10.1101/537860

    Gli1 astrocytes are distributed in a laminar-specific manner. (A) Tamoxifen-induced reporter expression in Gli1 CreER ; Ai14 mice reveals a laminar distribution of cells undergoing active Shh signaling (red). Scale bar, 250 μm. (B) Gli1+ cells (red) are a subset of all astrocytes (S100β, green, see inset; scale bar, 12.5 μm). (C) Layer analysis of Gli1 astrocytes (red) with DAPI and the deep layer marker Ctip2 (gray) illustrate their layer-specific organization. Scale bar, 125 μm. (D) The fraction of astrocytes expressing Gli1 in each cortical layer. Data points indicate individual animals, bars represent mean ± SEM. Statistical signficance was assessed by one-way ANOVA with Tukey’s post hoc test for multiple comparisons. *, p
    Figure Legend Snippet: Gli1 astrocytes are distributed in a laminar-specific manner. (A) Tamoxifen-induced reporter expression in Gli1 CreER ; Ai14 mice reveals a laminar distribution of cells undergoing active Shh signaling (red). Scale bar, 250 μm. (B) Gli1+ cells (red) are a subset of all astrocytes (S100β, green, see inset; scale bar, 12.5 μm). (C) Layer analysis of Gli1 astrocytes (red) with DAPI and the deep layer marker Ctip2 (gray) illustrate their layer-specific organization. Scale bar, 125 μm. (D) The fraction of astrocytes expressing Gli1 in each cortical layer. Data points indicate individual animals, bars represent mean ± SEM. Statistical signficance was assessed by one-way ANOVA with Tukey’s post hoc test for multiple comparisons. *, p

    Techniques Used: Expressing, Mouse Assay, Marker

    4) Product Images from "Resident c-kit+ cells in the heart are not cardiac stem cells"

    Article Title: Resident c-kit+ cells in the heart are not cardiac stem cells

    Journal: Nature Communications

    doi: 10.1038/ncomms9701

    Active c-kit endothelial expression and myogenic potential assayed by transient induction of Cre activity in c-kit MerCreMer/+ mice. ( a ) Diagram of the c-kit MerCreMer/+ allele . c-kit MerCreMer/+ animals were crossed to the ROSA26R tdTomato reporter line to obtain c-kit MerCreMer/+ ; ROSA26R tdTomato/+ . ( b – e ) Cre activity was transiently induced in c-kit MerCreMer/+ ; ROSA26R tdTomato/+ animals at P30, P60 and P90 by tamoxifen injection on days 1–3. Hearts were harvested on days 4 and 14. Many tdTomato + cells (arrows in b2, d2 and e2) were detected in hearts at P34 (b1), P64 (d1) and P104 (e1). These tdTomato + cells were PECAM + (c2, arrows, P30→34). b2, d2 and e2 are high-magnification florescent images of the areas outlined in b1, d1 and e1 (bright field), respectively. ( f ) Diagram of the cTnT nlacZ-H2B-GFP/+ allele and lineage tracing using c-kit MerCreMer/+ ; cTnT nlacZ-H2B-GFP/+ mice. Cre activity was transiently induced by tamoxifen injection for 4 days on days 1, 2, 3 and 5 (days 1 and 2 for E11.5). Samples were collected on day 7 (day 3 for E11.5). ( g ) cTnT H2B-GFP cells were detected at E13.5, P37, P67 and P97 (arrows), with the total number in the whole heart noted at the upper right corner. Scale bar, 1 mm (black) and 100 μm (white).
    Figure Legend Snippet: Active c-kit endothelial expression and myogenic potential assayed by transient induction of Cre activity in c-kit MerCreMer/+ mice. ( a ) Diagram of the c-kit MerCreMer/+ allele . c-kit MerCreMer/+ animals were crossed to the ROSA26R tdTomato reporter line to obtain c-kit MerCreMer/+ ; ROSA26R tdTomato/+ . ( b – e ) Cre activity was transiently induced in c-kit MerCreMer/+ ; ROSA26R tdTomato/+ animals at P30, P60 and P90 by tamoxifen injection on days 1–3. Hearts were harvested on days 4 and 14. Many tdTomato + cells (arrows in b2, d2 and e2) were detected in hearts at P34 (b1), P64 (d1) and P104 (e1). These tdTomato + cells were PECAM + (c2, arrows, P30→34). b2, d2 and e2 are high-magnification florescent images of the areas outlined in b1, d1 and e1 (bright field), respectively. ( f ) Diagram of the cTnT nlacZ-H2B-GFP/+ allele and lineage tracing using c-kit MerCreMer/+ ; cTnT nlacZ-H2B-GFP/+ mice. Cre activity was transiently induced by tamoxifen injection for 4 days on days 1, 2, 3 and 5 (days 1 and 2 for E11.5). Samples were collected on day 7 (day 3 for E11.5). ( g ) cTnT H2B-GFP cells were detected at E13.5, P37, P67 and P97 (arrows), with the total number in the whole heart noted at the upper right corner. Scale bar, 1 mm (black) and 100 μm (white).

    Techniques Used: Expressing, Activity Assay, Mouse Assay, Injection

    Cell type and lineage of c-kit + cells in the injured heart. ( a ) c-kit H2B-GFP -positive cells were present in the infarcted region of Tie2 Cre ; c-kit nlacZ-H2B-GFP/+ hearts at 30 dps. a2 is green channel of a1, and a3 is high-magnification image of the area outlined in a2. ( b ) Masson trichrome staining of cTnT MerCreMer/+ ; c-kit nlacZ-H2B-GFP/+ ; ROSA26R tdTomato/+ hearts at 60 dps shows the infarcted region. ( c ) Adjacent section of b . ROSA26R tdTomato signal indicates myocardial cells after tamoxifen induction (c1). No c-kit H2B-GFP cells were observed in the infarcted zone (arrows). c2 is green channel of c1. ( d ) Masson trichrome staining of c-kit MerCreMer/+ ; cTnT nlacZ-H2B-GFP/+ hearts at 60 dps. ( e ) Adjacent section of d shows a few cTnT H2B-GFP cells (
    Figure Legend Snippet: Cell type and lineage of c-kit + cells in the injured heart. ( a ) c-kit H2B-GFP -positive cells were present in the infarcted region of Tie2 Cre ; c-kit nlacZ-H2B-GFP/+ hearts at 30 dps. a2 is green channel of a1, and a3 is high-magnification image of the area outlined in a2. ( b ) Masson trichrome staining of cTnT MerCreMer/+ ; c-kit nlacZ-H2B-GFP/+ ; ROSA26R tdTomato/+ hearts at 60 dps shows the infarcted region. ( c ) Adjacent section of b . ROSA26R tdTomato signal indicates myocardial cells after tamoxifen induction (c1). No c-kit H2B-GFP cells were observed in the infarcted zone (arrows). c2 is green channel of c1. ( d ) Masson trichrome staining of c-kit MerCreMer/+ ; cTnT nlacZ-H2B-GFP/+ hearts at 60 dps. ( e ) Adjacent section of d shows a few cTnT H2B-GFP cells (

    Techniques Used: Staining

    5) Product Images from "An Immunocompetent Mouse Model of HPV16(+) Head and Neck Squamous Cell Carcinoma"

    Article Title: An Immunocompetent Mouse Model of HPV16(+) Head and Neck Squamous Cell Carcinoma

    Journal: Cell reports

    doi: 10.1016/j.celrep.2019.10.005

    Generation and Application of Inducible Reporter Knockin Mice (iLumiFluor) for Developing Methods to Target Lingual and Oropharyngeal Epithelia (A) General strategy for testing the efficacy of intralingual (i.l.) tamoxifen (TAM) administration to activate the estrogen receptor Cre recombinase fusion (KRT14-CreER tam ) and induce eGFP-NanoLuc (GpNLuc LumiFluor) transgene expression within the tongue following Cre-mediated recombination and excision of the STOP cassette. (B) Schematic of transverse and sagittal views highlighting key lingual anatomic features including the oropharynx, dorsal tongue, and ventral tongue. TAM was delivered by i.l. injection to the posterior, dorsal-lateral tongue to minimize dorsal tongue swelling and disruption to eating or drinking. (C) Targeted induction of bioluminescent signal in adult tongues. Bioluminescent imaging (BLI) of iLumiFluor and control littermate mice injected either 2× or 3× i.l. versus 5× i.p. with TAM. Live animal, non-invasive BLI was performed temporally and signal levels graphed relative to baseline. Representative images for both live, non-invasive BLI and end-point resected tongues 16 days post-TAM are presented (n = 3, ***p
    Figure Legend Snippet: Generation and Application of Inducible Reporter Knockin Mice (iLumiFluor) for Developing Methods to Target Lingual and Oropharyngeal Epithelia (A) General strategy for testing the efficacy of intralingual (i.l.) tamoxifen (TAM) administration to activate the estrogen receptor Cre recombinase fusion (KRT14-CreER tam ) and induce eGFP-NanoLuc (GpNLuc LumiFluor) transgene expression within the tongue following Cre-mediated recombination and excision of the STOP cassette. (B) Schematic of transverse and sagittal views highlighting key lingual anatomic features including the oropharynx, dorsal tongue, and ventral tongue. TAM was delivered by i.l. injection to the posterior, dorsal-lateral tongue to minimize dorsal tongue swelling and disruption to eating or drinking. (C) Targeted induction of bioluminescent signal in adult tongues. Bioluminescent imaging (BLI) of iLumiFluor and control littermate mice injected either 2× or 3× i.l. versus 5× i.p. with TAM. Live animal, non-invasive BLI was performed temporally and signal levels graphed relative to baseline. Representative images for both live, non-invasive BLI and end-point resected tongues 16 days post-TAM are presented (n = 3, ***p

    Techniques Used: Knock-In, Mouse Assay, Expressing, Injection, Imaging

    Autochthonous HPV16 E6 and E7 Coupled with PIK3CA E545K Expression Induces Preneoplastic Lesions That Rapidly Progress to Squamous Cell Carcinoma (A) General strategy for generating conditional and inducible iKHP mice by crossing the iKH strain to an inducible PIK3CA E545K strain (P). Representative photographs of independent tumors from iKHP animals at 8 weeks. (B) Mutant PIK3CA cooperates with HPV16 E6 and E7 oncogenes to drive tumorigenesis. R epresentative images of the gross morphology for control and iKHP tongues at various times post-tamoxifen (left). Representative H E-stained sections of control tongues compared to iKHP tongues harboring early, pre-malignant lesions (4–6 weeks) or tumors (6–8 weeks; middle). Representative invasive squamous cancer displaying both papillary, exophytic growth, and ulcerative lesion with basement membrane disruption, muscle invasion, and poorly differentiated areas. Tumor inset highlights invasive features (right, 2003) with mitotic figures, nuclear pleomorphism, and hyperchromatism (yellow arrowheads) and keratin pearls (black arrowheads). Magnification (left), 40×; bar, 200 µm. (C) Quantification of epidermal versus dermal Cd8a+ cytotoxic T cell infiltration (left) or FoxP3+ regulatory T cell infiltration (right) in control versus pre-malignant lesions or frank tumors (n = 3, mean ± SEM; ***p
    Figure Legend Snippet: Autochthonous HPV16 E6 and E7 Coupled with PIK3CA E545K Expression Induces Preneoplastic Lesions That Rapidly Progress to Squamous Cell Carcinoma (A) General strategy for generating conditional and inducible iKHP mice by crossing the iKH strain to an inducible PIK3CA E545K strain (P). Representative photographs of independent tumors from iKHP animals at 8 weeks. (B) Mutant PIK3CA cooperates with HPV16 E6 and E7 oncogenes to drive tumorigenesis. R epresentative images of the gross morphology for control and iKHP tongues at various times post-tamoxifen (left). Representative H E-stained sections of control tongues compared to iKHP tongues harboring early, pre-malignant lesions (4–6 weeks) or tumors (6–8 weeks; middle). Representative invasive squamous cancer displaying both papillary, exophytic growth, and ulcerative lesion with basement membrane disruption, muscle invasion, and poorly differentiated areas. Tumor inset highlights invasive features (right, 2003) with mitotic figures, nuclear pleomorphism, and hyperchromatism (yellow arrowheads) and keratin pearls (black arrowheads). Magnification (left), 40×; bar, 200 µm. (C) Quantification of epidermal versus dermal Cd8a+ cytotoxic T cell infiltration (left) or FoxP3+ regulatory T cell infiltration (right) in control versus pre-malignant lesions or frank tumors (n = 3, mean ± SEM; ***p

    Techniques Used: Expressing, Mouse Assay, Mutagenesis, Staining

    Targeted E6 and E7 Expression Induces Hyperplasia and Dysplasia with Reduced Off-Target Effects (A) General strategy for generating conditional and inducible iKH mice by crossing the KRT14-CreER tam (iK) strain to our knockin HPV16 strain (H). (B) Representative photographs of iKH animals at 8–10 months. (C) Comparison of oral volume fold change between LH, KH, and iKH and control littermate mice 42 weeks post-tamoxifen injection reveals that restricting expression to the lingual and oropharyngeal epithelia significantly reduces off-target effects on external oral epithelial volume (n = 3, *p
    Figure Legend Snippet: Targeted E6 and E7 Expression Induces Hyperplasia and Dysplasia with Reduced Off-Target Effects (A) General strategy for generating conditional and inducible iKH mice by crossing the KRT14-CreER tam (iK) strain to our knockin HPV16 strain (H). (B) Representative photographs of iKH animals at 8–10 months. (C) Comparison of oral volume fold change between LH, KH, and iKH and control littermate mice 42 weeks post-tamoxifen injection reveals that restricting expression to the lingual and oropharyngeal epithelia significantly reduces off-target effects on external oral epithelial volume (n = 3, *p

    Techniques Used: Expressing, Mouse Assay, Knock-In, Injection

    6) Product Images from "Off-Target Effects in Transgenic Mice: Characterization of Dopamine Transporter (DAT)-Cre Transgenic Mouse Lines Exposes Multiple Non-Dopaminergic Neuronal Clusters Available for Selective Targeting within Limbic Neurocircuitry"

    Article Title: Off-Target Effects in Transgenic Mice: Characterization of Dopamine Transporter (DAT)-Cre Transgenic Mouse Lines Exposes Multiple Non-Dopaminergic Neuronal Clusters Available for Selective Targeting within Limbic Neurocircuitry

    Journal: eNeuro

    doi: 10.1523/ENEURO.0198-19.2019

    Histological analyses using double- in situ hybridization of tdTom and Dat or Th mRNA and immunofluorescence of tdTOM show selective recombination in the ventral midbrain using constitutive and tamoxifen-inducible DAT-Cre mouse lines and identifies ectopic tdTOM-positive neurons in the amygdala. tdTom mRNA in coronal sections of ventral midbrain of tdTom DAT-Cre ( A ) and ( B ) tdTom DAT-CreERT2 mice 10 weeks of age. C , D , Double-FISH detecting tdTomato (red) and Dat (green) mRNA in the SNc and subregions of the VTA (IF, PBP, PN, and RLi) of the midbrain. Dat/tdTom mRNA overlap in the SNc and PBP shown in yellow (scale bar 500 μm). Higher magnification of insets of ( C’–C’’’ ) tdTom DAT-Cre and ( D’–D’’’ ) tdTom DAT-CreERT2 mice (scale bar: 25 μm). E , F , Double-FISH for tdTom (red) and Th (green) mRNA in the SNc and VTA with co-localization shown in yellow (scale bar: 500 μm). Higher magnification of insets of ( E’–E’’’ ) tdTom DAT-Cre and ( F’–F’’’ ) tdTom DAT-CreERT2 (scale bar: 25 μm). G , Immunofluorescent coronal section of ventral midbrain showing tdTOM in the SN and VTA but also in BLP (scale bar: 500 μm). tdTom, tdTomato mRNA; tdTOM, tdTomato protein; CA3, CA3 region of hippocampus; BLP, Basolateral amygdaloid nucleus posterior part; Dat, Dopamine transporter; IF, Interfascicular nucleus; PBP, Parabrachial pigmented area; PN, Paranigral nuclei; RLi, Rostral linear nucleus; SNc, Substantia nigra pars compacta ; SNr, Substantia nigra pars reticulata ; tdTom, tdTomato mRNA; tdTOM, tdTomato protein; Th, Tyrosine hydroxylase; VTA: Ventral tegmental area; CA3, CA3 region of hippocampus.
    Figure Legend Snippet: Histological analyses using double- in situ hybridization of tdTom and Dat or Th mRNA and immunofluorescence of tdTOM show selective recombination in the ventral midbrain using constitutive and tamoxifen-inducible DAT-Cre mouse lines and identifies ectopic tdTOM-positive neurons in the amygdala. tdTom mRNA in coronal sections of ventral midbrain of tdTom DAT-Cre ( A ) and ( B ) tdTom DAT-CreERT2 mice 10 weeks of age. C , D , Double-FISH detecting tdTomato (red) and Dat (green) mRNA in the SNc and subregions of the VTA (IF, PBP, PN, and RLi) of the midbrain. Dat/tdTom mRNA overlap in the SNc and PBP shown in yellow (scale bar 500 μm). Higher magnification of insets of ( C’–C’’’ ) tdTom DAT-Cre and ( D’–D’’’ ) tdTom DAT-CreERT2 mice (scale bar: 25 μm). E , F , Double-FISH for tdTom (red) and Th (green) mRNA in the SNc and VTA with co-localization shown in yellow (scale bar: 500 μm). Higher magnification of insets of ( E’–E’’’ ) tdTom DAT-Cre and ( F’–F’’’ ) tdTom DAT-CreERT2 (scale bar: 25 μm). G , Immunofluorescent coronal section of ventral midbrain showing tdTOM in the SN and VTA but also in BLP (scale bar: 500 μm). tdTom, tdTomato mRNA; tdTOM, tdTomato protein; CA3, CA3 region of hippocampus; BLP, Basolateral amygdaloid nucleus posterior part; Dat, Dopamine transporter; IF, Interfascicular nucleus; PBP, Parabrachial pigmented area; PN, Paranigral nuclei; RLi, Rostral linear nucleus; SNc, Substantia nigra pars compacta ; SNr, Substantia nigra pars reticulata ; tdTom, tdTomato mRNA; tdTOM, tdTomato protein; Th, Tyrosine hydroxylase; VTA: Ventral tegmental area; CA3, CA3 region of hippocampus.

    Techniques Used: In Situ Hybridization, Immunofluorescence, Mouse Assay, Fluorescence In Situ Hybridization

    7) Product Images from "Evidence That Dendritic Mitochondria Negatively Regulate Dendritic Branching in Pyramidal Neurons in the Neocortex"

    Article Title: Evidence That Dendritic Mitochondria Negatively Regulate Dendritic Branching in Pyramidal Neurons in the Neocortex

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.5095-13.2014

    Overexpression of TRAK2-MBD alters mitochondrial distribution and dendritic morphology. A–D , Pyramidal neurons expressing EGFP and mito-RFP in P7.5 cortices electroporated in utero with a control mock vector ( A , B ) or TRAK2-MBD-expression vector ( C , D ) at E13.5–13.75. Expressions of TRAK2-MBD and EGFP were induced at E18.5 by tamoxifen administration. The red dotted lines indicate the boundary between layers 1 and 2. The regions indicated by the red brackets are enlarged in A′–D′ . A′ - D′ , Enlarged images of the proximal portion of the apical dendrite in control ( A′ , B′ ) and TRAK2-MBD neurons ( C′ , D′ ). In the TRAK2-MBD neuron, mito-RFP signals (red) are reduced in dendrites and noticeably more branches emanate from the proximal portion of the dendrites. B″ , D″ , Enlarged images of the mito-RFP signals in the somata of control ( B″ ) and TRAK2-MBD neurons ( D″ ) show no overfused mitochondria. E , F , Neurolucida drawings of the control ( E ) and TRAK2-MBD neurons ( F ). Note the increase in dendritic branching in TRAK2-MBD neurons. G – I , Analysis of the dendritic morphology: ( G ) the respective number of dendritic branch points of apical and basal dendrites and their sum, ( H ) the lengths of apical and basal dendrites and their sum, and ( I ) the number of branch points in the apical dendrite relative to the distance from the soma; n = 10 cells, five brains for control; 18 cells, six brains for TRAK2-MBD. J – M , Pyramidal neurons expressing EGFP and mito-RFP in P21.5 cortices. No obvious alterations are seen in the dendritic morphology or mitochondrial distribution in the TRAK2-MBD neuron. Scale bars: A–D , 20 μm; A′–D′ , 10 μm; B″ , D″ , 5 μm; E , F , 50 μm; J–M , 40 μm. Error bars indicate SEM; ** p
    Figure Legend Snippet: Overexpression of TRAK2-MBD alters mitochondrial distribution and dendritic morphology. A–D , Pyramidal neurons expressing EGFP and mito-RFP in P7.5 cortices electroporated in utero with a control mock vector ( A , B ) or TRAK2-MBD-expression vector ( C , D ) at E13.5–13.75. Expressions of TRAK2-MBD and EGFP were induced at E18.5 by tamoxifen administration. The red dotted lines indicate the boundary between layers 1 and 2. The regions indicated by the red brackets are enlarged in A′–D′ . A′ - D′ , Enlarged images of the proximal portion of the apical dendrite in control ( A′ , B′ ) and TRAK2-MBD neurons ( C′ , D′ ). In the TRAK2-MBD neuron, mito-RFP signals (red) are reduced in dendrites and noticeably more branches emanate from the proximal portion of the dendrites. B″ , D″ , Enlarged images of the mito-RFP signals in the somata of control ( B″ ) and TRAK2-MBD neurons ( D″ ) show no overfused mitochondria. E , F , Neurolucida drawings of the control ( E ) and TRAK2-MBD neurons ( F ). Note the increase in dendritic branching in TRAK2-MBD neurons. G – I , Analysis of the dendritic morphology: ( G ) the respective number of dendritic branch points of apical and basal dendrites and their sum, ( H ) the lengths of apical and basal dendrites and their sum, and ( I ) the number of branch points in the apical dendrite relative to the distance from the soma; n = 10 cells, five brains for control; 18 cells, six brains for TRAK2-MBD. J – M , Pyramidal neurons expressing EGFP and mito-RFP in P21.5 cortices. No obvious alterations are seen in the dendritic morphology or mitochondrial distribution in the TRAK2-MBD neuron. Scale bars: A–D , 20 μm; A′–D′ , 10 μm; B″ , D″ , 5 μm; E , F , 50 μm; J–M , 40 μm. Error bars indicate SEM; ** p

    Techniques Used: Over Expression, Expressing, In Utero, Plasmid Preparation

    8) Product Images from "RUNX1 marks a luminal castration resistant lineage established at the onset of prostate development"

    Article Title: RUNX1 marks a luminal castration resistant lineage established at the onset of prostate development

    Journal: bioRxiv

    doi: 10.1101/2020.06.19.161604

    RUNX1 + cells contribute to the establishment of the proximal luminal lineage during embryonic prostate development. ( A ) Strategy for lineage-tracing of RUNX1 + cells in UGS explant cultures. Tamoxifen was applied on day 0 and day 1 and washed out on day 2. ( B, C ) Co-immunostaining of RFP, RUNX1, CDH1 in UGS explants harvested at day 2 ( B ) and day 7 ( C ). Higher magnification images of proximal (i) and (ii) distal regions are shown for day 7. Arrows show RFp + RUNX1-low cells, arrowheads show RFP + RUNX1 + cells. Scale bars: 200μm (yellow) and 50μm (white). ( C ) Quantification of % of epithelial RUNX1 + cells in the RFP subset at day 2 ( n = 7) and day 7 ( n = 3) of UGS explant cultures. Quantification was performed within the boundaries delimited in B by dotted lines. ( E, F ) Co-immunostaining of RFP, NKX3.1, CDH1 in UGS explants harvested at day 2 ( E ) and day 7 ( F ). Higher magnification images of (i) proximal and (ii) distal regions are shown for day 7. Arrows show RFP + NKX3.1 + cells, arrowheads show RFP + NKX3.1 - cells. Scale bars: 200μm (yellow) and 50μm (white). ( G ) Quantification of % of epithelial NKX3.1 + cells in the RFP subset at day 2 ( n = 6) and day 7 ( n = 4) of UGS explant cultures. Quantification was performed within the boundaries delimited in F by dotted lines. ( H, I ) Coimmunostaining of RFP, K4, CDH1 in UGS explants harvested at day 2 ( H ) and day 7 ( I ). Higher magnification images of (i) proximal and (ii) distal regions are shown for day 7. Arrows show RFP + K4 - cells, arrowheads show RFP + K4 + cells. Scale bars: 200μm (yellow) and 50μm (white). ( J ) Quantification of % of epithelial K4 + cells in the RFP subset at day 2 ( n = 3) and day 7 ( n = 3) of UGS explant cultures. Quantification was performed within the boundaries delimited in I by dotted lines.
    Figure Legend Snippet: RUNX1 + cells contribute to the establishment of the proximal luminal lineage during embryonic prostate development. ( A ) Strategy for lineage-tracing of RUNX1 + cells in UGS explant cultures. Tamoxifen was applied on day 0 and day 1 and washed out on day 2. ( B, C ) Co-immunostaining of RFP, RUNX1, CDH1 in UGS explants harvested at day 2 ( B ) and day 7 ( C ). Higher magnification images of proximal (i) and (ii) distal regions are shown for day 7. Arrows show RFp + RUNX1-low cells, arrowheads show RFP + RUNX1 + cells. Scale bars: 200μm (yellow) and 50μm (white). ( C ) Quantification of % of epithelial RUNX1 + cells in the RFP subset at day 2 ( n = 7) and day 7 ( n = 3) of UGS explant cultures. Quantification was performed within the boundaries delimited in B by dotted lines. ( E, F ) Co-immunostaining of RFP, NKX3.1, CDH1 in UGS explants harvested at day 2 ( E ) and day 7 ( F ). Higher magnification images of (i) proximal and (ii) distal regions are shown for day 7. Arrows show RFP + NKX3.1 + cells, arrowheads show RFP + NKX3.1 - cells. Scale bars: 200μm (yellow) and 50μm (white). ( G ) Quantification of % of epithelial NKX3.1 + cells in the RFP subset at day 2 ( n = 6) and day 7 ( n = 4) of UGS explant cultures. Quantification was performed within the boundaries delimited in F by dotted lines. ( H, I ) Coimmunostaining of RFP, K4, CDH1 in UGS explants harvested at day 2 ( H ) and day 7 ( I ). Higher magnification images of (i) proximal and (ii) distal regions are shown for day 7. Arrows show RFP + K4 - cells, arrowheads show RFP + K4 + cells. Scale bars: 200μm (yellow) and 50μm (white). ( J ) Quantification of % of epithelial K4 + cells in the RFP subset at day 2 ( n = 3) and day 7 ( n = 3) of UGS explant cultures. Quantification was performed within the boundaries delimited in I by dotted lines.

    Techniques Used: Immunostaining

    Lineage tracing of RUNX1 expressing cells labelled in intact mice. ( A ) Scheme of the tamoxifen labelling strategy to evaluate the labelling efficiency of Runx1 expressing cells. ( B ) Flow-cytometry analysis of intact ( n = 2), castrated ( n = 2), and regenerated ( n = 2) Runx1 CRE-stop-RFP mice, and corresponding quantification of the percentage of RFP + cells in the epithelial EPCAM + fraction. ( C ) Estimated absolute number of epithelial RFP + cells in intact ( n = 5) and castrated ( n = 5) and regenerated ( n = 4) prostates based on IHC quantifications. Int: Intact, Cas: Castrated, Rgn: Regenerated. ( D ) Quantification of % of epithelial RFP + cells being K4 + or K4 - in intact ( n = 3), castrated ( n = 4) and regenerated ( n = 5) mice. ( F ) Coimmunostaining showing the enrichment of K4 + RFP labelled cells in the proximal region of the AP (top) and DLVP (bottom) prostate lobes. Scale bar: 50μm.
    Figure Legend Snippet: Lineage tracing of RUNX1 expressing cells labelled in intact mice. ( A ) Scheme of the tamoxifen labelling strategy to evaluate the labelling efficiency of Runx1 expressing cells. ( B ) Flow-cytometry analysis of intact ( n = 2), castrated ( n = 2), and regenerated ( n = 2) Runx1 CRE-stop-RFP mice, and corresponding quantification of the percentage of RFP + cells in the epithelial EPCAM + fraction. ( C ) Estimated absolute number of epithelial RFP + cells in intact ( n = 5) and castrated ( n = 5) and regenerated ( n = 4) prostates based on IHC quantifications. Int: Intact, Cas: Castrated, Rgn: Regenerated. ( D ) Quantification of % of epithelial RFP + cells being K4 + or K4 - in intact ( n = 3), castrated ( n = 4) and regenerated ( n = 5) mice. ( F ) Coimmunostaining showing the enrichment of K4 + RFP labelled cells in the proximal region of the AP (top) and DLVP (bottom) prostate lobes. Scale bar: 50μm.

    Techniques Used: Expressing, Mouse Assay, Flow Cytometry, Immunohistochemistry

    9) Product Images from "Wnt-mediated interactions of tumor-initiating cells with a macrophage niche drive skin tumor formation"

    Article Title: Wnt-mediated interactions of tumor-initiating cells with a macrophage niche drive skin tumor formation

    Journal: bioRxiv

    doi: 10.1101/2020.07.17.209338

    Loss of Wnt from tSCs and macrophages leads to tumor regression. (A) Protocol of Tamoxifen or/and RU486 administration in mice with DMBA/TPA-induced skin lesions. See Methods for more details. (B) Tumor size 8 weeks after RU467, TAM or RU486+TAM administration in Bulge ΔWls (n = 9), Mac ΔWls (n = 9), and Bulge ΔWls Mac ΔWls (n = 3) mice and controls (n = 12, 12, 4), respectively. (C) FACS quantification of CD34 + CD49f + tSCs in Bulge ΔWls (n = 6), Mac ΔWls (n = 6) and Bulge ΔWls Mac ΔWls (n = 4) mice and control (n = 8,7,3) mice, respectively (Left). Quantification of SOX2 + immunostained tSC numbers by IF in skin lesions (n = 3 mice in each group) (Right). (D) FACS quantification of CD34 + CD49f + TCF/Lef:H2B/GFP + tSCs in Bulge ΔWls (n = 6), Mac ΔWls (n = 6) and Bulge Mac ΔWls (n = 3) mice and control (n = 8, 8, 8) mice, respectively. (E) FACS quantification of CD45 + CD11b + F4/80 + TCF/Lef:H2B/GFP + TAMs in Bulge ΔWls (n = 13), Mac ΔWls (n = 6) and Bulge ΔWls Mac ΔWls (n = 3) mice and control (n = 8, 8, 8) mice, respectively. ( F ) Immunostaining for SOX2 + and F4/80 + cells in Bulge DWls and Mac DWls mouse skin at the last point of tumor regression. DAPI nuclear staining is represented in blue. Scale bar 20 μm. (B) Mann-Whitney test. (C), (D) , (E) and (G) . Unpaired two-tailed Student’s t -test. All error bars represent s.e.m. *P
    Figure Legend Snippet: Loss of Wnt from tSCs and macrophages leads to tumor regression. (A) Protocol of Tamoxifen or/and RU486 administration in mice with DMBA/TPA-induced skin lesions. See Methods for more details. (B) Tumor size 8 weeks after RU467, TAM or RU486+TAM administration in Bulge ΔWls (n = 9), Mac ΔWls (n = 9), and Bulge ΔWls Mac ΔWls (n = 3) mice and controls (n = 12, 12, 4), respectively. (C) FACS quantification of CD34 + CD49f + tSCs in Bulge ΔWls (n = 6), Mac ΔWls (n = 6) and Bulge ΔWls Mac ΔWls (n = 4) mice and control (n = 8,7,3) mice, respectively (Left). Quantification of SOX2 + immunostained tSC numbers by IF in skin lesions (n = 3 mice in each group) (Right). (D) FACS quantification of CD34 + CD49f + TCF/Lef:H2B/GFP + tSCs in Bulge ΔWls (n = 6), Mac ΔWls (n = 6) and Bulge Mac ΔWls (n = 3) mice and control (n = 8, 8, 8) mice, respectively. (E) FACS quantification of CD45 + CD11b + F4/80 + TCF/Lef:H2B/GFP + TAMs in Bulge ΔWls (n = 13), Mac ΔWls (n = 6) and Bulge ΔWls Mac ΔWls (n = 3) mice and control (n = 8, 8, 8) mice, respectively. ( F ) Immunostaining for SOX2 + and F4/80 + cells in Bulge DWls and Mac DWls mouse skin at the last point of tumor regression. DAPI nuclear staining is represented in blue. Scale bar 20 μm. (B) Mann-Whitney test. (C), (D) , (E) and (G) . Unpaired two-tailed Student’s t -test. All error bars represent s.e.m. *P

    Techniques Used: Mouse Assay, FACS, Immunostaining, Staining, MANN-WHITNEY, Two Tailed Test

    10) Product Images from "Suppression of Tumor Growth and Angiogenesis by a Specific Antagonist of the Cell-Surface Expressed Nucleolin"

    Article Title: Suppression of Tumor Growth and Angiogenesis by a Specific Antagonist of the Cell-Surface Expressed Nucleolin

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0002518

    HB-19 inhibits tumor growth in the nude mice. (A) HB-19 inhibits the growth of MDA-MB-231 tumor-cell xenografts. Cells (2×10 6 ) were injected subcutaneously into the right flank of female nude mice. Two weeks later, mice with a palpable tumor of approximately 40 mm 3 in volume were randomly separated into three groups (n = 5) and were given peritumoral injections 3 times/week of 0.1 ml PBS alone (Control), HB-19 (5 mg/kg), or Tamoxifen (Tmx) 10 mg/kg) for 6 weeks. (B) HB-19 inhibits the growth of MDA-MB-435 tumor-cell xenografts. Cells (1×10 6 ) were injected in the mammary fat pad of female nude mice. Two weeks later, mice with a palpable tumor were randomly separated into three groups (n = 10) and were given intraperitoneal injections 3 times/week of 0.1 ml PBS alone (Control), HB-19 (5 mg/kg), or 5-fluouracil (5-FU, 40 mg/kg) for 8 weeks. At the end of each experiment (in A and B), mice were sacrificed and the tumors were excised and weighed. The results are presented as the mean weight ±standard deviation (±S.D.) obtained from the number of mice in each group. (C, D) Inhibition of tumor development in mice treated by intraperitoneal (i.p.) and subcutaneous (s.c.) administration of HB-19. MDA-MB-231 tumor bearing mice in three groups (n = 10) were treated with HB-19 (10 mg/kg) by i.p. or s.c. injections, 3 times/week for 28 days. The arrow at day 0 shows initiation of HB-19 treatment. Panel D shows MDA-MB-231 tumor bearing mice, untreated control and HB-19 treated (i.p. injection). Statistical significance: *p
    Figure Legend Snippet: HB-19 inhibits tumor growth in the nude mice. (A) HB-19 inhibits the growth of MDA-MB-231 tumor-cell xenografts. Cells (2×10 6 ) were injected subcutaneously into the right flank of female nude mice. Two weeks later, mice with a palpable tumor of approximately 40 mm 3 in volume were randomly separated into three groups (n = 5) and were given peritumoral injections 3 times/week of 0.1 ml PBS alone (Control), HB-19 (5 mg/kg), or Tamoxifen (Tmx) 10 mg/kg) for 6 weeks. (B) HB-19 inhibits the growth of MDA-MB-435 tumor-cell xenografts. Cells (1×10 6 ) were injected in the mammary fat pad of female nude mice. Two weeks later, mice with a palpable tumor were randomly separated into three groups (n = 10) and were given intraperitoneal injections 3 times/week of 0.1 ml PBS alone (Control), HB-19 (5 mg/kg), or 5-fluouracil (5-FU, 40 mg/kg) for 8 weeks. At the end of each experiment (in A and B), mice were sacrificed and the tumors were excised and weighed. The results are presented as the mean weight ±standard deviation (±S.D.) obtained from the number of mice in each group. (C, D) Inhibition of tumor development in mice treated by intraperitoneal (i.p.) and subcutaneous (s.c.) administration of HB-19. MDA-MB-231 tumor bearing mice in three groups (n = 10) were treated with HB-19 (10 mg/kg) by i.p. or s.c. injections, 3 times/week for 28 days. The arrow at day 0 shows initiation of HB-19 treatment. Panel D shows MDA-MB-231 tumor bearing mice, untreated control and HB-19 treated (i.p. injection). Statistical significance: *p

    Techniques Used: Mouse Assay, Multiple Displacement Amplification, Injection, Standard Deviation, Inhibition

    11) Product Images from "Tamoxifen Provides Structural and Functional Rescue in Murine Models of Photoreceptor Degeneration"

    Article Title: Tamoxifen Provides Structural and Functional Rescue in Murine Models of Photoreceptor Degeneration

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.2717-16.2017

    Tamoxifen administration reduced microglial activation and infiltration induced by acute LI. A , In vivo fundus autofluorescence (FAF) imaging after LI revealed the emergence of a punctate hyperautofluorescent pattern centered in the superotemporal quandrant
    Figure Legend Snippet: Tamoxifen administration reduced microglial activation and infiltration induced by acute LI. A , In vivo fundus autofluorescence (FAF) imaging after LI revealed the emergence of a punctate hyperautofluorescent pattern centered in the superotemporal quandrant

    Techniques Used: Activation Assay, In Vivo, Imaging

    Tamoxifen administration suppresses microglial activation and inflammatory cytokine production and reduces microglial toxicity to photoreceptors. A , Effect of tamoxifen (TMX) on retinal microglia activation and inflammatory cytokine expression was assessed
    Figure Legend Snippet: Tamoxifen administration suppresses microglial activation and inflammatory cytokine production and reduces microglial toxicity to photoreceptors. A , Effect of tamoxifen (TMX) on retinal microglia activation and inflammatory cytokine expression was assessed

    Techniques Used: Activation Assay, Expressing

    Tamoxifen-mediated rescue of photoreceptor degeneration in LI model is not dependent on the sex of experimental animals. Young adult (2–3 months old) female and male mice were subjected to LI with and without pretreatment with tamoxifen. OCT evaluation
    Figure Legend Snippet: Tamoxifen-mediated rescue of photoreceptor degeneration in LI model is not dependent on the sex of experimental animals. Young adult (2–3 months old) female and male mice were subjected to LI with and without pretreatment with tamoxifen. OCT evaluation

    Techniques Used: Mouse Assay

    Tamoxifen administration before acute LI results in near complete rescue of structural and functional retinal damage. Adult mice were administered daily oral tamoxifen (added to standard animal chow, 500 mg/kg chow, estimated intake of 80 mg/kg body weight/d)
    Figure Legend Snippet: Tamoxifen administration before acute LI results in near complete rescue of structural and functional retinal damage. Adult mice were administered daily oral tamoxifen (added to standard animal chow, 500 mg/kg chow, estimated intake of 80 mg/kg body weight/d)

    Techniques Used: Functional Assay, Mouse Assay

    Evaluation of the effects of tamoxifen on photoreceptor survival and function. A , Measurement of ERG recovery after a moderate bleaching flash in animals pretreated for oral tamoxifen for 7 d compared with age-matched control fed a standard control diet.
    Figure Legend Snippet: Evaluation of the effects of tamoxifen on photoreceptor survival and function. A , Measurement of ERG recovery after a moderate bleaching flash in animals pretreated for oral tamoxifen for 7 d compared with age-matched control fed a standard control diet.

    Techniques Used:

    Tamoxifen administration provides structural and functional rescue of photoreceptor degeneration in rd10 mouse model for RP. rd10 mice were provided either standard chow (control group) or tamoxifen-supplemented chow from P21 and evaluated at different
    Figure Legend Snippet: Tamoxifen administration provides structural and functional rescue of photoreceptor degeneration in rd10 mouse model for RP. rd10 mice were provided either standard chow (control group) or tamoxifen-supplemented chow from P21 and evaluated at different

    Techniques Used: Functional Assay, Mouse Assay

    12) Product Images from "Selective inhibition of Ebola entry with selective estrogen receptor modulators by disrupting the endolysosomal calcium"

    Article Title: Selective inhibition of Ebola entry with selective estrogen receptor modulators by disrupting the endolysosomal calcium

    Journal: Scientific Reports

    doi: 10.1038/srep41226

    SERMs reduce the cellular sphingosine. ( a ) Standard curves for quantification of Sph (0.1–100 ng/mL) with C17-Sph (10 ng/mL) as the internal standard. ( b) Measurement of changes in intracellular levels of Sph in HepG2 cells. Cells were treated with 10 μM tamoxifen, clomiphene or U18666a for 1 h, then lipids were extracted and the levels of Sph were analyzed. Data are expressed as the means ± SEM (n = 3). Significant differences versus control group are presented by asterisks (*), *** P
    Figure Legend Snippet: SERMs reduce the cellular sphingosine. ( a ) Standard curves for quantification of Sph (0.1–100 ng/mL) with C17-Sph (10 ng/mL) as the internal standard. ( b) Measurement of changes in intracellular levels of Sph in HepG2 cells. Cells were treated with 10 μM tamoxifen, clomiphene or U18666a for 1 h, then lipids were extracted and the levels of Sph were analyzed. Data are expressed as the means ± SEM (n = 3). Significant differences versus control group are presented by asterisks (*), *** P

    Techniques Used:

    Equal dosages of SERMs inhibit Ebola pseudovirion entry and induce cholesterol accumulation. ( a ) Dose-response inhibition of Ebola entry for tamoxifen, clomiphene and U18666a. Data are expressed as the means ± SEM (n = 3). ( b ) Dose-response cholesterol accumulation for tamoxifen, clomiphene and U18666a. ( b ) Time-response cholesterol accumulation for tamoxifen, clomiphene and U18666a. Black arrows indicate examples of cholesterol accumulation. ( d,e ) The quantification of cholesterol accumulation stained with filipin. Data are expressed as the means ± SEM (n ≥ 4). Significant differences versus control group (0 μM or 0 h) are presented by asterisks (*), * P
    Figure Legend Snippet: Equal dosages of SERMs inhibit Ebola pseudovirion entry and induce cholesterol accumulation. ( a ) Dose-response inhibition of Ebola entry for tamoxifen, clomiphene and U18666a. Data are expressed as the means ± SEM (n = 3). ( b ) Dose-response cholesterol accumulation for tamoxifen, clomiphene and U18666a. ( b ) Time-response cholesterol accumulation for tamoxifen, clomiphene and U18666a. Black arrows indicate examples of cholesterol accumulation. ( d,e ) The quantification of cholesterol accumulation stained with filipin. Data are expressed as the means ± SEM (n ≥ 4). Significant differences versus control group (0 μM or 0 h) are presented by asterisks (*), * P

    Techniques Used: Inhibition, Staining

    SERMs upregulate the endolysosomal calcium levels. ( a ) Representative images of the endolysosomal calcium release of HepG2 detected by Fluo8-AM calcium indicator at 1 s, 60 s, 90 s, 330 s, 390 s, and 420 s after incubation of 10 μM tamoxifen, clomiphene or U18666a for 1 h. ( b ) The calcium release of HepG2 loaded with 1 μM TG at 60 s for 30 s and 200 μM GPN at 330 s for 60 s. HepG2 cells were pre-treated with 10 μM tamoxifen, clomiphene or U18666a for 1 h. ( c ) The quantification of calcium release induced by 200 μM GPN. Data are expressed as the means ± SEM (n > 20). Significant differences versus control group are presented by asterisks (*), *** P
    Figure Legend Snippet: SERMs upregulate the endolysosomal calcium levels. ( a ) Representative images of the endolysosomal calcium release of HepG2 detected by Fluo8-AM calcium indicator at 1 s, 60 s, 90 s, 330 s, 390 s, and 420 s after incubation of 10 μM tamoxifen, clomiphene or U18666a for 1 h. ( b ) The calcium release of HepG2 loaded with 1 μM TG at 60 s for 30 s and 200 μM GPN at 330 s for 60 s. HepG2 cells were pre-treated with 10 μM tamoxifen, clomiphene or U18666a for 1 h. ( c ) The quantification of calcium release induced by 200 μM GPN. Data are expressed as the means ± SEM (n > 20). Significant differences versus control group are presented by asterisks (*), *** P

    Techniques Used: Incubation

    SERMs selectively inhibit the entry of Ebola pseudovirion, but not through the estrogen receptor pathway. ( a ) Effects of E 2 , ICI, tamoxifen, and clomiphene on Ebola/WSN/VSV pseudovirion entry. HepG2 cells were pretreated with 10 nM E2, 100 nM ICI, 10 μM tamoxifen, and 10 μM clomiphene for 1 h and infected by Ebola/WSN/VSV pseudovirion with compounds for 24 h. The cells were then lysed, and the luciferase assay was carried out. NH 4 Cl serves as a negative control. ( b ) Representative images of colocalization of VLPs (red, marked by mCheery-VP40) with TPC2 (green, marked with EGFP) and LAMP1 (blue, marked with BFP) from 10 μM tamoxifen or 10 μM clomiphene treated HepG2. White arrows indicate examples of colocalization. Data are expressed as the means ± SEM (n = 3). Significant differences versus control group are presented by asterisks (*), ** P
    Figure Legend Snippet: SERMs selectively inhibit the entry of Ebola pseudovirion, but not through the estrogen receptor pathway. ( a ) Effects of E 2 , ICI, tamoxifen, and clomiphene on Ebola/WSN/VSV pseudovirion entry. HepG2 cells were pretreated with 10 nM E2, 100 nM ICI, 10 μM tamoxifen, and 10 μM clomiphene for 1 h and infected by Ebola/WSN/VSV pseudovirion with compounds for 24 h. The cells were then lysed, and the luciferase assay was carried out. NH 4 Cl serves as a negative control. ( b ) Representative images of colocalization of VLPs (red, marked by mCheery-VP40) with TPC2 (green, marked with EGFP) and LAMP1 (blue, marked with BFP) from 10 μM tamoxifen or 10 μM clomiphene treated HepG2. White arrows indicate examples of colocalization. Data are expressed as the means ± SEM (n = 3). Significant differences versus control group are presented by asterisks (*), ** P

    Techniques Used: Infection, Luciferase, Negative Control

    13) Product Images from "Loss of endogenous Nfatc1 reduces the rate of DMBA/TPA-induced skin tumorigenesis"

    Article Title: Loss of endogenous Nfatc1 reduces the rate of DMBA/TPA-induced skin tumorigenesis

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E15-05-0282

    Nfatc1 deletion decreases the rate of tumor initiation but not tumor promotion. (A) Schematic of inducible K19 -specific Nfatc1 deletion. (B) Nfatc1 immunostaining (green) in iKO mice 5 d after tamoxifen/vehicle. (C) DMBA/TPA initiation (ODT) and promotion (DOT) regimes. (D, G) Percentage of tumor-free iKO/control mice in (D) ODT or (G) DOT regime ( n = 16 mice/genotype). (E, H) Average tumor number during (E) ODT or (H) DOT regime. Data: mean ± SEM (seven mice/genotype). Several time points were significantly different (*); mixed-effect model. (F, I) Tumor formation rate during (F) ODT regime is significantly different but not during (I) DOT regime (mixed-effect model; time: continuous variable). (J) Real-time PCR of Nfatc1 in tumor cells relative to FACS-sorted bulge cells. Data: mean ± SD (six mice/group). Immunostaining for (K) CD45 (red) and (L) Tcrδ (red) in cKO/control tumors 8–10 wk post-DMBA. (M) Quantification of Tcrδ+ cells in cKO/control tumors. Data: mean ± SEM (three mice/genotype). DAPI, blue; dotted line, epithelial–stromal barrier; white dots, hair shaft autofluorescence. Scale bar, 25 μm (B), 50 μm (K, L).
    Figure Legend Snippet: Nfatc1 deletion decreases the rate of tumor initiation but not tumor promotion. (A) Schematic of inducible K19 -specific Nfatc1 deletion. (B) Nfatc1 immunostaining (green) in iKO mice 5 d after tamoxifen/vehicle. (C) DMBA/TPA initiation (ODT) and promotion (DOT) regimes. (D, G) Percentage of tumor-free iKO/control mice in (D) ODT or (G) DOT regime ( n = 16 mice/genotype). (E, H) Average tumor number during (E) ODT or (H) DOT regime. Data: mean ± SEM (seven mice/genotype). Several time points were significantly different (*); mixed-effect model. (F, I) Tumor formation rate during (F) ODT regime is significantly different but not during (I) DOT regime (mixed-effect model; time: continuous variable). (J) Real-time PCR of Nfatc1 in tumor cells relative to FACS-sorted bulge cells. Data: mean ± SD (six mice/group). Immunostaining for (K) CD45 (red) and (L) Tcrδ (red) in cKO/control tumors 8–10 wk post-DMBA. (M) Quantification of Tcrδ+ cells in cKO/control tumors. Data: mean ± SEM (three mice/genotype). DAPI, blue; dotted line, epithelial–stromal barrier; white dots, hair shaft autofluorescence. Scale bar, 25 μm (B), 50 μm (K, L).

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

    Nfatc1 deletion decreases stem cell contribution to papillomas during tumor initiation. (A) Schematic of inducible K19 -specific Nfatc1 deletion and mTmG labeling. (B) Representative images of K19 CreERT; mTmG follicles after tamoxifen. Images show unlabeled (only mT+ cells) or labeled (containing mG+ cells) follicles. (C) Percentage of mG+ hair follicles 8–12 wk post-DMBA (initiation, ODT; promotion, DOT). Data are mean ± SEM for 50–100 hair follicles from four individual mice/group. (D) Representative sagittal section of a tumor containing mG+ cells derived from K19 -expressing bulge cells. (E) Percentage of tumors containing mG+ cells in iKO mice and littermate controls from sagittally sectioned tumors during ODT and DOT regimes. Data are mean ± SEM of three or four individual mice/genotype for 8–15 tumors from each mouse. Tumors were harvested 8–12 wk post-DMBA. DAPI, blue. White dots denote hair shaft autofluorescence. Scale bar, 25 μm.
    Figure Legend Snippet: Nfatc1 deletion decreases stem cell contribution to papillomas during tumor initiation. (A) Schematic of inducible K19 -specific Nfatc1 deletion and mTmG labeling. (B) Representative images of K19 CreERT; mTmG follicles after tamoxifen. Images show unlabeled (only mT+ cells) or labeled (containing mG+ cells) follicles. (C) Percentage of mG+ hair follicles 8–12 wk post-DMBA (initiation, ODT; promotion, DOT). Data are mean ± SEM for 50–100 hair follicles from four individual mice/group. (D) Representative sagittal section of a tumor containing mG+ cells derived from K19 -expressing bulge cells. (E) Percentage of tumors containing mG+ cells in iKO mice and littermate controls from sagittally sectioned tumors during ODT and DOT regimes. Data are mean ± SEM of three or four individual mice/genotype for 8–15 tumors from each mouse. Tumors were harvested 8–12 wk post-DMBA. DAPI, blue. White dots denote hair shaft autofluorescence. Scale bar, 25 μm.

    Techniques Used: Labeling, Mouse Assay, Derivative Assay, Expressing

    14) Product Images from "UFBP1, a Key Component of the Ufm1 Conjugation System, Is Essential for Ufmylation-Mediated Regulation of Erythroid Development"

    Article Title: UFBP1, a Key Component of the Ufm1 Conjugation System, Is Essential for Ufmylation-Mediated Regulation of Erythroid Development

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1005643

    Loss of UFBP1 in adult mice results in severe pancytopenia and animal death. a . Confirmation of UFBP1 depletion in bone marrow cells of TAM-injected CKO mice. Floxed mice were IP injected with tamoxifen according to a standard protocol. BM cells were collected and subject to immunoblotting of UFBP1. b. Survival curve of UFBP1 deficient mice after TAM injection, p
    Figure Legend Snippet: Loss of UFBP1 in adult mice results in severe pancytopenia and animal death. a . Confirmation of UFBP1 depletion in bone marrow cells of TAM-injected CKO mice. Floxed mice were IP injected with tamoxifen according to a standard protocol. BM cells were collected and subject to immunoblotting of UFBP1. b. Survival curve of UFBP1 deficient mice after TAM injection, p

    Techniques Used: Mouse Assay, Injection

    15) Product Images from "Type I Interferon Receptor Signaling of Neurons and Astrocytes Regulates Microglia Activation during Viral Encephalitis"

    Article Title: Type I Interferon Receptor Signaling of Neurons and Astrocytes Regulates Microglia Activation during Viral Encephalitis

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2018.09.003

    Myeloid Cell Subsets within the OB of Infected Animals Consist of CD45 hi Microglia and Show Distinct Gene Expression Profiles Tamoxifen-treated CX3CR1-cre- ER+/− td-tomato St/Wt mice were treated as described in Figure 1 . On 6 dpi, mice were perfused and immune cells from OB were isolated. CD45 low CD11b low microglia from control animals (MG ctrl.) and CD45 low CD11b low microglia (CD45 low MG inf.), CD45 hi CD11b hi td-tomato + microglia (CD45 hi MG inf.), and CD45 hi CD11b hi td-tomato − Ly6G − infiltrating monocytes (Mon.) from infected animals were FACS sorted. (A) Flow cytometry data of immune cells isolated from OB. Three OBs were pooled for control animals, and five OBs were pooled for VSV-infected animals (representative data, N = 2). (B) Expression analysis for most regulated genes from the different myeloid cell populations between control and VSV-infected animals. (C) Principal-component analysis of different myeloid cell populations. (D) Venn diagram describing numbers of common and different genes regulated between indicated populations compared to CD45 low CD11b low microglia of control animals. (E) mRNA expression levels of the indicated genes in different myeloid cell populations.
    Figure Legend Snippet: Myeloid Cell Subsets within the OB of Infected Animals Consist of CD45 hi Microglia and Show Distinct Gene Expression Profiles Tamoxifen-treated CX3CR1-cre- ER+/− td-tomato St/Wt mice were treated as described in Figure 1 . On 6 dpi, mice were perfused and immune cells from OB were isolated. CD45 low CD11b low microglia from control animals (MG ctrl.) and CD45 low CD11b low microglia (CD45 low MG inf.), CD45 hi CD11b hi td-tomato + microglia (CD45 hi MG inf.), and CD45 hi CD11b hi td-tomato − Ly6G − infiltrating monocytes (Mon.) from infected animals were FACS sorted. (A) Flow cytometry data of immune cells isolated from OB. Three OBs were pooled for control animals, and five OBs were pooled for VSV-infected animals (representative data, N = 2). (B) Expression analysis for most regulated genes from the different myeloid cell populations between control and VSV-infected animals. (C) Principal-component analysis of different myeloid cell populations. (D) Venn diagram describing numbers of common and different genes regulated between indicated populations compared to CD45 low CD11b low microglia of control animals. (E) mRNA expression levels of the indicated genes in different myeloid cell populations.

    Techniques Used: Infection, Expressing, Mouse Assay, Isolation, FACS, Flow Cytometry

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

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

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201511061

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

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

    17) Product Images from "Synergistic epigenetic reactivation of estrogen receptor-? (ER?) by combined green tea polyphenol and histone deacetylase inhibitor in ER?-negative breast cancer cells"

    Article Title: Synergistic epigenetic reactivation of estrogen receptor-? (ER?) by combined green tea polyphenol and histone deacetylase inhibitor in ER?-negative breast cancer cells

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-9-274

    Treatment with EGCG and TSA retrieved responsiveness to E 2 and tamoxifen in ER-negative cells . A) Cellular viability in response to E 2 and tamoxifen. B) The expression of PGR, an ERα target gene, in response to E 2 and tamoxifen. EGCG and/or TSA-pretreated MDA-MB-231 cells were treated with or without 10 nM of E 2 or 1 μM tamoxifen for 2 days. MCF-7 cells served as a positive control. Cells were harvested at the indicated time periods and assessed for cellular viability and PGR expression, respectively. Cellular viability was measured by Trypan blue exclusion assay. PGR expression was detected by quantitative real-time PCR. Data were obtained from three independent experiments and normalized to GAPDH and calibrated to levels in samples without treatment of E 2 and tamoxifen. Bars, SD; *, P
    Figure Legend Snippet: Treatment with EGCG and TSA retrieved responsiveness to E 2 and tamoxifen in ER-negative cells . A) Cellular viability in response to E 2 and tamoxifen. B) The expression of PGR, an ERα target gene, in response to E 2 and tamoxifen. EGCG and/or TSA-pretreated MDA-MB-231 cells were treated with or without 10 nM of E 2 or 1 μM tamoxifen for 2 days. MCF-7 cells served as a positive control. Cells were harvested at the indicated time periods and assessed for cellular viability and PGR expression, respectively. Cellular viability was measured by Trypan blue exclusion assay. PGR expression was detected by quantitative real-time PCR. Data were obtained from three independent experiments and normalized to GAPDH and calibrated to levels in samples without treatment of E 2 and tamoxifen. Bars, SD; *, P

    Techniques Used: Expressing, Multiple Displacement Amplification, Positive Control, Trypan Blue Exclusion Assay, Real-time Polymerase Chain Reaction

    18) Product Images from "Epidermal Growth Factor Receptor-Dependent Regulation of Integrin-Mediated Signaling and Cell Cycle Entry in Epithelial Cells"

    Article Title: Epidermal Growth Factor Receptor-Dependent Regulation of Integrin-Mediated Signaling and Cell Cycle Entry in Epithelial Cells

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.19.8586-8599.2004

    EGFR and Myc overexpression rescues S phase. (A) CV1 cells were serum starved for 48 h and either left in suspension (S) or plated on FN in the absence (FN) or presence (EGF) of 40 ng of EGF/ml for the indicated times. The levels of cyclin D1 (cycD1), p27, cyclin A (cycA), and Rb phosphorylation at Thr821 (P-Rb T821) were monitored by immunoblotting with the respective antibodies. Total levels of Akt were monitored to control for total protein levels. (B) CV1 cells (CV) were infected with retroviruses expressing EGFR (R1, R5) or cyclin D1 (D1) or with an empty vector (V). Levels of EGFR and cyclin D1 expression were monitored by immunoblotting of whole-cell extracts with their respective antibodies after plating of cells on FN for 12 h. Levels of EGFR activation in virus-infected cells held in suspension or attached to FN were monitored by immunoblotting of EGFR immunoprecipitates with anti-phosphotyrosine antibodies (P-Tyr Blot). Total levels of EGFR were monitored by immunoblotting with an anti-EGFR antibody (EGFR Blot). *, the length of film exposure to enhanced chemiluminescence was 5 times less for EGFR-overexpressing cells than for other samples. (C) Adherent CV1 cells infected with retroviral vectors encoding either EGFR, cyclin D1, or no cDNA (Vector) were serum starved for 48 h and either placed in suspension or plated on FN for the indicated times. Levels of cyclin A and p27 were monitored by immunoblotting cell lysates with their respective antibodies. Rb phosphorylation at Thr821 was monitored by immunoblotting of cell lysates with phosphospecific antibodies. Total levels of Akt were monitored by immunoblotting as a control for total protein levels in the extracts. (D) CV1 cells were serum starved for 48 h and either placed in suspension or plated on FN in the absence or presence of 40 ng of EGF/ml for the indicated times. Adherent CV1 cells infected with retroviral vectors encoding EGFR or no cDNA were serum starved for 48 h and then either placed in suspension or plated on FN for the indicated times. Levels of Myc expression were monitored by immunoblotting of cell lysates with anti-Myc antibodies. Total levels of Akt were monitored by immunoblotting as a control for total protein levels in the extracts. (E) Adherent CV1 cells infected with retroviral vectors encoding either EGFR or no cDNA were serum starved for 48 h and then either placed in suspension or plated on FN for the indicated times. Levels of cyclin D1 were monitored by immunoblotting of cell lysates with anti-cyclin D1 antibodies. Erk and Akt activation was monitored by immunoblotting of cell lysates with phosphospecific antibodies (P-Erk, P-Akt). Total levels of Erk and Akt were monitored by immunoblotting as a control for total protein levels in the extracts (Erk, Akt). (F) CV1 cells were infected with a virus containing an empty vector or expressing MycER. Levels of MycER expression were monitored by immunoblotting with anti-Myc antibodies after plating on FN in the absence (FN) or presence (Tx) of 50 nM tamoxifen. Levels of p27 and cyclin A expression in cells in suspension or at the indicated times after plating on FN in the presence or absence of 50 nM tamoxifen were monitored by immunoblotting. Total levels of Akt were monitored by immunoblotting as a control for total protein levels in the extracts.
    Figure Legend Snippet: EGFR and Myc overexpression rescues S phase. (A) CV1 cells were serum starved for 48 h and either left in suspension (S) or plated on FN in the absence (FN) or presence (EGF) of 40 ng of EGF/ml for the indicated times. The levels of cyclin D1 (cycD1), p27, cyclin A (cycA), and Rb phosphorylation at Thr821 (P-Rb T821) were monitored by immunoblotting with the respective antibodies. Total levels of Akt were monitored to control for total protein levels. (B) CV1 cells (CV) were infected with retroviruses expressing EGFR (R1, R5) or cyclin D1 (D1) or with an empty vector (V). Levels of EGFR and cyclin D1 expression were monitored by immunoblotting of whole-cell extracts with their respective antibodies after plating of cells on FN for 12 h. Levels of EGFR activation in virus-infected cells held in suspension or attached to FN were monitored by immunoblotting of EGFR immunoprecipitates with anti-phosphotyrosine antibodies (P-Tyr Blot). Total levels of EGFR were monitored by immunoblotting with an anti-EGFR antibody (EGFR Blot). *, the length of film exposure to enhanced chemiluminescence was 5 times less for EGFR-overexpressing cells than for other samples. (C) Adherent CV1 cells infected with retroviral vectors encoding either EGFR, cyclin D1, or no cDNA (Vector) were serum starved for 48 h and either placed in suspension or plated on FN for the indicated times. Levels of cyclin A and p27 were monitored by immunoblotting cell lysates with their respective antibodies. Rb phosphorylation at Thr821 was monitored by immunoblotting of cell lysates with phosphospecific antibodies. Total levels of Akt were monitored by immunoblotting as a control for total protein levels in the extracts. (D) CV1 cells were serum starved for 48 h and either placed in suspension or plated on FN in the absence or presence of 40 ng of EGF/ml for the indicated times. Adherent CV1 cells infected with retroviral vectors encoding EGFR or no cDNA were serum starved for 48 h and then either placed in suspension or plated on FN for the indicated times. Levels of Myc expression were monitored by immunoblotting of cell lysates with anti-Myc antibodies. Total levels of Akt were monitored by immunoblotting as a control for total protein levels in the extracts. (E) Adherent CV1 cells infected with retroviral vectors encoding either EGFR or no cDNA were serum starved for 48 h and then either placed in suspension or plated on FN for the indicated times. Levels of cyclin D1 were monitored by immunoblotting of cell lysates with anti-cyclin D1 antibodies. Erk and Akt activation was monitored by immunoblotting of cell lysates with phosphospecific antibodies (P-Erk, P-Akt). Total levels of Erk and Akt were monitored by immunoblotting as a control for total protein levels in the extracts (Erk, Akt). (F) CV1 cells were infected with a virus containing an empty vector or expressing MycER. Levels of MycER expression were monitored by immunoblotting with anti-Myc antibodies after plating on FN in the absence (FN) or presence (Tx) of 50 nM tamoxifen. Levels of p27 and cyclin A expression in cells in suspension or at the indicated times after plating on FN in the presence or absence of 50 nM tamoxifen were monitored by immunoblotting. Total levels of Akt were monitored by immunoblotting as a control for total protein levels in the extracts.

    Techniques Used: Over Expression, Infection, Expressing, Plasmid Preparation, Activation Assay

    19) Product Images from "C-Cbl reverses HER2-mediated tamoxifen resistance in human breast cancer cells"

    Article Title: C-Cbl reverses HER2-mediated tamoxifen resistance in human breast cancer cells

    Journal: BMC Cancer

    doi: 10.1186/s12885-018-4387-5

    PP2 and trastuzumab inhibit the formation of ER-c-Src-HER2 and partially restore tamoxifen sensitivity. a BT474 cells were treated with PP2 (10 μM) for 4 h and subjected to immunoprecipitation and immunoblotting as indicated. b MTT assays in BT474 cells treated with PP2 (10 μM) for 4 h and then treated with 17β-E2 (10 nmol/L) and/or tamoxifen (1 μmol/L) for 48 h. * P
    Figure Legend Snippet: PP2 and trastuzumab inhibit the formation of ER-c-Src-HER2 and partially restore tamoxifen sensitivity. a BT474 cells were treated with PP2 (10 μM) for 4 h and subjected to immunoprecipitation and immunoblotting as indicated. b MTT assays in BT474 cells treated with PP2 (10 μM) for 4 h and then treated with 17β-E2 (10 nmol/L) and/or tamoxifen (1 μmol/L) for 48 h. * P

    Techniques Used: Immunoprecipitation, MTT Assay

    Overexpression of c-Cbl 70Z in BT474 cells. a BT474 cells were transfected with PSVL-70Z-c-Cbl (OE-70Zc-Cbl) or PSVL vector (Vector) and c-Cbl level was examined by immunoblot analysis. b BT474 cells were transfected with PSVL vector (Vec) or PSVL-70Zc-Cbl (70Z) for 48 h and then examined by immunoprecipitation and immunoblot analysis. c BT474 cells were transfected with PSVL-70Zc-Cbl for 24 h and then treated with vehicle, estrogen (10 nmol/L), or tamoxifen (1 μmol/L) for another 48 h. Total viable cell number was measured by MTT assays. Data represent the average of three independent replicates ± SD. * P
    Figure Legend Snippet: Overexpression of c-Cbl 70Z in BT474 cells. a BT474 cells were transfected with PSVL-70Z-c-Cbl (OE-70Zc-Cbl) or PSVL vector (Vector) and c-Cbl level was examined by immunoblot analysis. b BT474 cells were transfected with PSVL vector (Vec) or PSVL-70Zc-Cbl (70Z) for 48 h and then examined by immunoprecipitation and immunoblot analysis. c BT474 cells were transfected with PSVL-70Zc-Cbl for 24 h and then treated with vehicle, estrogen (10 nmol/L), or tamoxifen (1 μmol/L) for another 48 h. Total viable cell number was measured by MTT assays. Data represent the average of three independent replicates ± SD. * P

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Immunoprecipitation, MTT Assay

    Inhibition of lipid rafts suppresses the formation of the ER-c-Src-HER2 complex and partially restores tamoxifen sensitivity. a BT474 cells were treated with vehicle (Con) or nystatin (N) (5 μg/ml) for 2 h and subjected to immunoprecipitation and immunoblotting analysis as indicated. b Cells were seeded in 6-well plates (2.5 × 10 4 cells/well) for 24 h and then treated with vehicle or nystatin(5 μg/ml) for 2 h, followed by treatment with vehicle, E2(10 nmol/L), TAM(1 μmol/L), or the combination for 4 h. c Cells were seeded in 96-well plates (5000 cells/well). At 24 h after plating, cells were treated with vehicle or nystatin (5 μg/ml) for 2 h, and then treated with vehicle, E2(10 nmol/L), TAM(1 μmol/L), or the combination. After 48 h, MTT assays were performed. The bar graph shows the sensitivity of BT474 cells to tamoxifen before and after treatment with nystatin. Data represent the mean ± SD of at least three independent experiments. P values were determined by the Student’s t test. (* p
    Figure Legend Snippet: Inhibition of lipid rafts suppresses the formation of the ER-c-Src-HER2 complex and partially restores tamoxifen sensitivity. a BT474 cells were treated with vehicle (Con) or nystatin (N) (5 μg/ml) for 2 h and subjected to immunoprecipitation and immunoblotting analysis as indicated. b Cells were seeded in 6-well plates (2.5 × 10 4 cells/well) for 24 h and then treated with vehicle or nystatin(5 μg/ml) for 2 h, followed by treatment with vehicle, E2(10 nmol/L), TAM(1 μmol/L), or the combination for 4 h. c Cells were seeded in 96-well plates (5000 cells/well). At 24 h after plating, cells were treated with vehicle or nystatin (5 μg/ml) for 2 h, and then treated with vehicle, E2(10 nmol/L), TAM(1 μmol/L), or the combination. After 48 h, MTT assays were performed. The bar graph shows the sensitivity of BT474 cells to tamoxifen before and after treatment with nystatin. Data represent the mean ± SD of at least three independent experiments. P values were determined by the Student’s t test. (* p

    Techniques Used: Inhibition, Immunoprecipitation, MTT Assay

    Proposed model for the role of c-Cbl in tamoxifen resistance of HER2-overexpressing breast cancer cells. In HER2-overexpressing breast cancer cells, ER-c-Src-HER2 complex formation results in HER2 signaling pathway activation and tamoxifen resistance. c-Cbl reduces ER-c-Src-HER2 complex formation by inhibiting lipid rafts and restores the sensitivity to tamoxifen
    Figure Legend Snippet: Proposed model for the role of c-Cbl in tamoxifen resistance of HER2-overexpressing breast cancer cells. In HER2-overexpressing breast cancer cells, ER-c-Src-HER2 complex formation results in HER2 signaling pathway activation and tamoxifen resistance. c-Cbl reduces ER-c-Src-HER2 complex formation by inhibiting lipid rafts and restores the sensitivity to tamoxifen

    Techniques Used: Activation Assay

    Overexpression of c-Cbl reverses HER2-mediated tamoxifen resistance. a c-Cbl protein level was detected in BT474 and T47D cells. And then, BT474 cells were transfected with 3 × flag-CMV-9-c-Cbl (OE-c-Cbl) or 3 × flag-CMV-9 vector(Vector), and then examined the c-Cbl level by immunoblot analysis. b Immunoprecipitation after overexpression c-Cbl 48 h in BT474. c BT474 cells were transfected with control vector or c-Cbl overexpression plasmids, 24 h later, followed by vehicle, estrogen(10 nmol/L), and tamoxifen (1 μmol/L) or combination treatment for 4 h. Cell lysates were examined by immunoblot analysis using the indicated antibodies. d BT474 cells were transfected with plasmids expressing c-Cbl for 24 h, and then exposed to vehicle, estrogen(10 nmol/L), or tamoxifen(1 μmol/L) treatment for another 48 h. Total viable cell number was measured by MTT assays. Data represent the average of three independent replicates ± SD. (* p
    Figure Legend Snippet: Overexpression of c-Cbl reverses HER2-mediated tamoxifen resistance. a c-Cbl protein level was detected in BT474 and T47D cells. And then, BT474 cells were transfected with 3 × flag-CMV-9-c-Cbl (OE-c-Cbl) or 3 × flag-CMV-9 vector(Vector), and then examined the c-Cbl level by immunoblot analysis. b Immunoprecipitation after overexpression c-Cbl 48 h in BT474. c BT474 cells were transfected with control vector or c-Cbl overexpression plasmids, 24 h later, followed by vehicle, estrogen(10 nmol/L), and tamoxifen (1 μmol/L) or combination treatment for 4 h. Cell lysates were examined by immunoblot analysis using the indicated antibodies. d BT474 cells were transfected with plasmids expressing c-Cbl for 24 h, and then exposed to vehicle, estrogen(10 nmol/L), or tamoxifen(1 μmol/L) treatment for another 48 h. Total viable cell number was measured by MTT assays. Data represent the average of three independent replicates ± SD. (* p

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Immunoprecipitation, Expressing, MTT Assay

    BT474 cells are resistant to tamoxifen. a Characterization of BT474 and T47D cells. Western blot analysis of ER, HER2, PR, and c-Src and their phosphorylation status in both cell lines, and Actin is as a loading control. b BT474 and T47D cells were seeded in 96-well plates at a density of 5000 cells/well and then treated with 4-OH tamoxifen (0–10 μmol/L). After 3 days, MTT assays were performed. c BT474 and T47D cells were seeded in 96-well plates and 24 h after plating, cells were treated with vehicle (CON), 17β-E2 (E2) (10 nmol/L), tamoxifen (TAM) (1 μmol/L), or combination (E2/TAM). After 48 h, MTT assays were performed. The bar graph shows the sensitivity of cells to the indicated treatments. Data represent the mean ± SD of at least three independent experiments. d Effect of tamoxifen on colony formation in BT474 cells. After 14 days, colonies in 6-well plates were imaged and quantified by counting the number of colonies. At least nine wells per treatment group were quantified per value reported. The results show the mean from three independent experiments. * P
    Figure Legend Snippet: BT474 cells are resistant to tamoxifen. a Characterization of BT474 and T47D cells. Western blot analysis of ER, HER2, PR, and c-Src and their phosphorylation status in both cell lines, and Actin is as a loading control. b BT474 and T47D cells were seeded in 96-well plates at a density of 5000 cells/well and then treated with 4-OH tamoxifen (0–10 μmol/L). After 3 days, MTT assays were performed. c BT474 and T47D cells were seeded in 96-well plates and 24 h after plating, cells were treated with vehicle (CON), 17β-E2 (E2) (10 nmol/L), tamoxifen (TAM) (1 μmol/L), or combination (E2/TAM). After 48 h, MTT assays were performed. The bar graph shows the sensitivity of cells to the indicated treatments. Data represent the mean ± SD of at least three independent experiments. d Effect of tamoxifen on colony formation in BT474 cells. After 14 days, colonies in 6-well plates were imaged and quantified by counting the number of colonies. At least nine wells per treatment group were quantified per value reported. The results show the mean from three independent experiments. * P

    Techniques Used: Western Blot, MTT Assay

    ER-c-Src-HER2 complex formation in the tamoxifen-resistant breast cancer cell line BT474. a BT474 or T47D cells (approximately 60% confluent) were treated with 17β-E2 (10 nmol/L) and/or TAM (1 μmol/L) or left untreated (vehicle control) for 4 h. Lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. b BT474 and ( c ) T47D cells were treated with 17β-E2 (10 nmol/L) and/or tamoxifen (TAM) (1 μmol/L) for 4 h, and then lysates were immunoprecipitated using a c-Src antibody or IgG and subjected to immunoblot analysis using the indicated antibodies
    Figure Legend Snippet: ER-c-Src-HER2 complex formation in the tamoxifen-resistant breast cancer cell line BT474. a BT474 or T47D cells (approximately 60% confluent) were treated with 17β-E2 (10 nmol/L) and/or TAM (1 μmol/L) or left untreated (vehicle control) for 4 h. Lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. b BT474 and ( c ) T47D cells were treated with 17β-E2 (10 nmol/L) and/or tamoxifen (TAM) (1 μmol/L) for 4 h, and then lysates were immunoprecipitated using a c-Src antibody or IgG and subjected to immunoblot analysis using the indicated antibodies

    Techniques Used: Immunoprecipitation

    Knockdown of HER2 expression in BT474 cells suppresses the formation of the ER-c-Src-HER2 complex and partially restores tamoxifen sensitivity. a Western blot analysis of BT474 cells transfected with siRNA targeting HER2. b BT474 cells transfected with HER2 siRNA were subjected to immunoprecipitation and immunoblotting as indicated. c Western blot analysis of phosphorylated HER2, c-Src, and ER in BT474 cells knocked down for HER2 expression for 48 h and then treated with 17β-E2 (10 nmol/L) and/or tamoxifen (TAM) (1 μmol/L) for 4 h. d MTT assays in BT474 cells transfected with siRNA targeting HER2 for 24 h and then treated with 17β-E2 (10 nmol/L) and/or tamoxifen (TAM) (1 μmol/L) for another 48 h. e Colony formation assays of BT474 cells transfected with siRNA targeting HER2 for 24 h and treated with 17β-E2 (10 nmol/L) and/or tamoxifen (TAM) (1 μmol/L) for 14d
    Figure Legend Snippet: Knockdown of HER2 expression in BT474 cells suppresses the formation of the ER-c-Src-HER2 complex and partially restores tamoxifen sensitivity. a Western blot analysis of BT474 cells transfected with siRNA targeting HER2. b BT474 cells transfected with HER2 siRNA were subjected to immunoprecipitation and immunoblotting as indicated. c Western blot analysis of phosphorylated HER2, c-Src, and ER in BT474 cells knocked down for HER2 expression for 48 h and then treated with 17β-E2 (10 nmol/L) and/or tamoxifen (TAM) (1 μmol/L) for 4 h. d MTT assays in BT474 cells transfected with siRNA targeting HER2 for 24 h and then treated with 17β-E2 (10 nmol/L) and/or tamoxifen (TAM) (1 μmol/L) for another 48 h. e Colony formation assays of BT474 cells transfected with siRNA targeting HER2 for 24 h and treated with 17β-E2 (10 nmol/L) and/or tamoxifen (TAM) (1 μmol/L) for 14d

    Techniques Used: Expressing, Western Blot, Transfection, Immunoprecipitation, MTT Assay

    Overexpression of HER2 leads to tamoxifen resistance in T47D cells. a T47D cells were transfected with HER2-pEGFP-N1 plasmid (OE-HER2) or pEGFP-N1 vector as a control (Vector). Immunoblot analysis was performed using the indicated antibodies. b T47D cells were transfected with the HER2 overexpression vector for 48 h, and immunoprecipitation and immunoblot analysis were performed with the indicated antibodies. c MTT assays in HER2-overexpressing T47D cells treated with 17β-E2 and/or tamoxifen. T47D cells were transfected with pEGFP-N1 vector or HER2-pEGFP-N1 (OE-HER2) for 24 h, and then treated with vehicle (CON), 17β-E2 (E2) (10 nmol/L), tamoxifen (TAM) (1 μmol/L) or combination treatment (E2/TAM) for another 48 h. MTT assays were then performed. * P
    Figure Legend Snippet: Overexpression of HER2 leads to tamoxifen resistance in T47D cells. a T47D cells were transfected with HER2-pEGFP-N1 plasmid (OE-HER2) or pEGFP-N1 vector as a control (Vector). Immunoblot analysis was performed using the indicated antibodies. b T47D cells were transfected with the HER2 overexpression vector for 48 h, and immunoprecipitation and immunoblot analysis were performed with the indicated antibodies. c MTT assays in HER2-overexpressing T47D cells treated with 17β-E2 and/or tamoxifen. T47D cells were transfected with pEGFP-N1 vector or HER2-pEGFP-N1 (OE-HER2) for 24 h, and then treated with vehicle (CON), 17β-E2 (E2) (10 nmol/L), tamoxifen (TAM) (1 μmol/L) or combination treatment (E2/TAM) for another 48 h. MTT assays were then performed. * P

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Immunoprecipitation, MTT Assay

    20) Product Images from "Numb regulates somatic cell lineage commitment during early gonadogenesis in mice"

    Article Title: Numb regulates somatic cell lineage commitment during early gonadogenesis in mice

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.149203

    Numb/Numbl mutant gonads have an irregular surface and interior pockets of undifferentiated cells. (A,B) At E14.5, both XY (A) and XX (B) Numb/Numbl ). In mutant XY gonads (D), germ cell (PECAM1 positive, green) and Sertoli cell (NR5A1 positive, red) numbers were reduced. Some regions retained VCAM1 (blue) and NR5A1 (red) positive interstitial cells. However, several domains in the mutant gonad were negative for all tested differentiation markers. (E,F) Control XX gonads (E) contained PECAM1-positive germ cells (green), interstitial and granulosa cells (VCAM1 positive, blue, and NR5A1 positive, red, respectively). Similar to XY gonads, mutant XX gonads (F) showed a significant reduction of germ cells and other differentiated cell types and the presence of regions negative for differentiation markers. (G-J) In control XY and XX gonads (G,I), LHX9 is restricted to undifferentiated cells in the CE. However, in mutants (H,J), LHX9-positive domains are present in the gonad interior. SOX9 and VCAM1 label Sertoli and interstitial cells in G,H; FOXL2 and DDX4 label granulosa and germ cells in I,J. Throughout the figures, ‘Mutant’ refers to gonads from Numb flox/flox ;Numbl −/− ;ROSA-CreER or Numb flox/flox ;Numbl +/− ;ROSA-CreER embryos injected with tamoxifen at E8.75. ‘Control’ refers to all other genotypes resulting from the cross in which no phenotype was evident. Scale bars: 50 µm (C-F); 100 µm (G-J).
    Figure Legend Snippet: Numb/Numbl mutant gonads have an irregular surface and interior pockets of undifferentiated cells. (A,B) At E14.5, both XY (A) and XX (B) Numb/Numbl ). In mutant XY gonads (D), germ cell (PECAM1 positive, green) and Sertoli cell (NR5A1 positive, red) numbers were reduced. Some regions retained VCAM1 (blue) and NR5A1 (red) positive interstitial cells. However, several domains in the mutant gonad were negative for all tested differentiation markers. (E,F) Control XX gonads (E) contained PECAM1-positive germ cells (green), interstitial and granulosa cells (VCAM1 positive, blue, and NR5A1 positive, red, respectively). Similar to XY gonads, mutant XX gonads (F) showed a significant reduction of germ cells and other differentiated cell types and the presence of regions negative for differentiation markers. (G-J) In control XY and XX gonads (G,I), LHX9 is restricted to undifferentiated cells in the CE. However, in mutants (H,J), LHX9-positive domains are present in the gonad interior. SOX9 and VCAM1 label Sertoli and interstitial cells in G,H; FOXL2 and DDX4 label granulosa and germ cells in I,J. Throughout the figures, ‘Mutant’ refers to gonads from Numb flox/flox ;Numbl −/− ;ROSA-CreER or Numb flox/flox ;Numbl +/− ;ROSA-CreER embryos injected with tamoxifen at E8.75. ‘Control’ refers to all other genotypes resulting from the cross in which no phenotype was evident. Scale bars: 50 µm (C-F); 100 µm (G-J).

    Techniques Used: Mutagenesis, Injection

    21) Product Images from "The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations"

    Article Title: The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1118857109

    Differential responses of Lgr5 + vs. Bmi1 + lineages to acute radiation injury. ( A and B ) Tamoxifen-mediated lineage trace of Lgr5-eGFP + progeny in Lgr5-eGFP-IRES-CreERT2; Rosa-YFP mouse duodenum demonstrates prolific lineage generation under homeostasis
    Figure Legend Snippet: Differential responses of Lgr5 + vs. Bmi1 + lineages to acute radiation injury. ( A and B ) Tamoxifen-mediated lineage trace of Lgr5-eGFP + progeny in Lgr5-eGFP-IRES-CreERT2; Rosa-YFP mouse duodenum demonstrates prolific lineage generation under homeostasis

    Techniques Used:

    Clonogenic culture of single FACS-isolated Bmi1-YFP + ISCs. ( A – D ) Single sorted Bmi1-YFP + cells marked by 1 or 2 d tamoxifen administration in Bmi1-CreER; Rosa-YFP mice form intestinal epithelial spheroids in clonogenic culture that demonstrate
    Figure Legend Snippet: Clonogenic culture of single FACS-isolated Bmi1-YFP + ISCs. ( A – D ) Single sorted Bmi1-YFP + cells marked by 1 or 2 d tamoxifen administration in Bmi1-CreER; Rosa-YFP mice form intestinal epithelial spheroids in clonogenic culture that demonstrate

    Techniques Used: FACS, Isolation, Mouse Assay

    22) Product Images from "Release of HER2 repression of trefoil factor 3 (TFF3) expression mediates trastuzumab resistance in HER2+/ER+ mammary carcinoma"

    Article Title: Release of HER2 repression of trefoil factor 3 (TFF3) expression mediates trastuzumab resistance in HER2+/ER+ mammary carcinoma

    Journal: Oncotarget

    doi: 10.18632/oncotarget.18431

    Activation of HER2 decreased TFF3 expression, while inhibition of HER2 increased TFF3 expression in BT474 cells partially in an ERα-independent manner ( A – C) Left , BT474 cells were treated with 500 ng/ml EGF or HRG for 24 and 48 hours respectively, in phenol-red free media supplemented with 10% charcoal-stripped FBS. (A–C) Right , BT474 cells were treated with 200 ng/ml EGF or HRG for 48 hours in phenol-red free media supplemented with 10% charcoal-stripped FBS in the presence of 100 nM 17β-estradiol. ( D – F ) BT474 cells were treated with 10 µg/ml trastuzumab for 48 hours in phenol-red free media supplemented with 10% charcoal-stripped FBS ± 100 nM 17β-estradiol. (A and D) TFF3 promoter luciferase activity was measured with Renilla luciferase activity as transfection control. TFF3 (B and E) mRNA and (C and F) protein levels were determined by qPCR and western blot respectively, with β-ACTIN as input control. The densitometric analyses of protein bands were performed using ImageJ. ( G and H ) BT474 cells were treated with 10 µg/ml trastuzumab ± 100 nM 17β-estradiol ± 1 µM Tamoxifen for 48 hours in phenol-red free media supplemented with 10% charcoal-stripped FBS. (G) ERE and (H) TFF3 promoter luciferase activities were measured with Renilla luciferase activity as transfection control. UT: untreated; E2: 17β-estradiol; Tras: trastuzumab; Tam: tamoxifen; ERE: estrogen response element. * p
    Figure Legend Snippet: Activation of HER2 decreased TFF3 expression, while inhibition of HER2 increased TFF3 expression in BT474 cells partially in an ERα-independent manner ( A – C) Left , BT474 cells were treated with 500 ng/ml EGF or HRG for 24 and 48 hours respectively, in phenol-red free media supplemented with 10% charcoal-stripped FBS. (A–C) Right , BT474 cells were treated with 200 ng/ml EGF or HRG for 48 hours in phenol-red free media supplemented with 10% charcoal-stripped FBS in the presence of 100 nM 17β-estradiol. ( D – F ) BT474 cells were treated with 10 µg/ml trastuzumab for 48 hours in phenol-red free media supplemented with 10% charcoal-stripped FBS ± 100 nM 17β-estradiol. (A and D) TFF3 promoter luciferase activity was measured with Renilla luciferase activity as transfection control. TFF3 (B and E) mRNA and (C and F) protein levels were determined by qPCR and western blot respectively, with β-ACTIN as input control. The densitometric analyses of protein bands were performed using ImageJ. ( G and H ) BT474 cells were treated with 10 µg/ml trastuzumab ± 100 nM 17β-estradiol ± 1 µM Tamoxifen for 48 hours in phenol-red free media supplemented with 10% charcoal-stripped FBS. (G) ERE and (H) TFF3 promoter luciferase activities were measured with Renilla luciferase activity as transfection control. UT: untreated; E2: 17β-estradiol; Tras: trastuzumab; Tam: tamoxifen; ERE: estrogen response element. * p

    Techniques Used: Activation Assay, Expressing, Inhibition, Luciferase, Activity Assay, Transfection, Real-time Polymerase Chain Reaction, Western Blot

    23) Product Images from "Loss of Fgfr1 in chondrocytes inhibits osteoarthritis by promoting autophagic activity in temporomandibular joint"

    Article Title: Loss of Fgfr1 in chondrocytes inhibits osteoarthritis by promoting autophagic activity in temporomandibular joint

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.002293

    IHC staining for FGFR1 was performed in TMJ condylar cartilage of WT mice ( A ) and nonsurgery mice ( B and C ) 2 and 6 weeks ( W ) after UAC surgery. D–F, FGFR1 expressions in condylar cartilage of 6-, 12-, and 18-month-old ( MO ) mice. The percentages of FGFR1 immunoreactive positive cells in UAC surgery ( G ) and aging ( H ) TMJ OA models were analyzed ( n = 5–6 per group). I, scheme of experiment: Fgfr1 f/f mice were crossed with Col2a1-CreER T2 mice to generate Fgfr1 f/f ;Col2a1-CreER T2 and control mice. J–M, representative TMJ sections from Col2a1-CreER T2 ; Rosa26 tdTomato mice showing the high Cre recombination. N, efficiency of Fgfr1 deletion in TMJ cartilage from Fgfr1 cKO mice after tamoxifen administration was examined by quantitative-PCR ( n = 3 per group). Scale bar, 50 μm ( A–F ), 100 μm ( J and K ), and 200 μm ( L ). Data are expressed as the percent expression relative to controls. Values represent mean (symbols) ± S.D. ( error bar ). p values between groups with * are less than 0.05.
    Figure Legend Snippet: IHC staining for FGFR1 was performed in TMJ condylar cartilage of WT mice ( A ) and nonsurgery mice ( B and C ) 2 and 6 weeks ( W ) after UAC surgery. D–F, FGFR1 expressions in condylar cartilage of 6-, 12-, and 18-month-old ( MO ) mice. The percentages of FGFR1 immunoreactive positive cells in UAC surgery ( G ) and aging ( H ) TMJ OA models were analyzed ( n = 5–6 per group). I, scheme of experiment: Fgfr1 f/f mice were crossed with Col2a1-CreER T2 mice to generate Fgfr1 f/f ;Col2a1-CreER T2 and control mice. J–M, representative TMJ sections from Col2a1-CreER T2 ; Rosa26 tdTomato mice showing the high Cre recombination. N, efficiency of Fgfr1 deletion in TMJ cartilage from Fgfr1 cKO mice after tamoxifen administration was examined by quantitative-PCR ( n = 3 per group). Scale bar, 50 μm ( A–F ), 100 μm ( J and K ), and 200 μm ( L ). Data are expressed as the percent expression relative to controls. Values represent mean (symbols) ± S.D. ( error bar ). p values between groups with * are less than 0.05.

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

    24) Product Images from "Vasculature-associated cells expressing nestin in developing bones encompass early cells in the osteoblast and endothelial lineage"

    Article Title: Vasculature-associated cells expressing nestin in developing bones encompass early cells in the osteoblast and endothelial lineage

    Journal: Developmental cell

    doi: 10.1016/j.devcel.2014.03.014

    Non-endothelial nestin + cells encompass early cells of the osteoblast lineage a–c. Nes- GFP; Col2-cre; R26R Tomato femur sections stained for CD31 and nuclei. E12.5, x200 confocal (a) (right panel: CD31 single-color) and E13.5, x200 (b), x630 confocal of dotted area (c). Arrows: CD31 − Nes + Tomato + cells, allowheads: CD31 + Nes + Tomato − cells. d. Nes- GFP; Col2-creER; R26R Tomato E13.5 femur sections stained for CD31 and nuclei. Pregnant mice received 2mg tamoxifen at E12.5. x200 confocal. e, f. Nes- GFP; Osx-creER; R26R Tomato E13.5 femur sections stained for CD31 and nuclei. Pregnant mice received 2mg tamoxifen at E12.5. x200 (e), x630 confocal of the osteogenic perichondrium (f) (lower panel: CD31 single-color). Arrows: CD31 − Nes + Tomato + cells, arrowheads: perivascular Tomato + cells. Green: EGFP, red: tdTomato, blue: Alexa633, gray: DAPI. Scale bars: 50μm (a, b, d, e), 20μm (c, f). g. Flow cytometry analysis of E13.5 Nes- GFP; Osx-creER; R26R Tomato limb cells (tamoxifen at E12.5) stained for CD45. CD45 − fraction gated for Tomato + , blue line: Nes- GFP − Tomato + limb cells (n=3, data represented as mean ± S.D.). h. Nes- GFP; Osx-creER; R26R Tomato E16.5 femur sections (tamoxifen at E13.5) stained for nuclei. Arrowheads: Tomato + cells disappearing from the osteogenic perichondrium. x100. Green: EGFP, red: tdTomato, blue: DAPI. i, j. Nes- GFP; Osx-creER; R26R Tomato E17.5 femur sections (tamoxifen at E16.5) stained for CD31 and nuclei. x100 (i), x630 confocal of dotted area (j). Arrows: CD31 − Nes + Tomato + cells on the innermost layer of the osteogenic perichondrium. Scale bars: 200μm (h, i), 50μm (j). k–m. Nes- GFP; Col2-creER; R26R Tomato P0 femur sections (tamoxifen at E13.5) stained for CD31 and nuclei. x100 (k), x630 confocal of dotted area (l: perichondrium, m: primary spongiosa). Arrows: CD31 − Nes + Tomato + cells. Green: EGFP, red: tdTomato, blue: Alexa633, gray: DAPI. Scale bars: 200μm (k), 20μm (l, m). o–q. E13.5 femur sections stained for CD31 and nuclei. Control (o), Ihh −/ − (p) and Runx2 −/ − (q), x200 confocal. Green: EGFP, red: Alexa546, gray: DAPI. Scale bars: 50μm. r. Quantification of CD31 + Nes + cells and CD31 − Nes + cells in perichondrium. Control (while bars) and Ihh −/ − (black bars), n=4 per group, *p
    Figure Legend Snippet: Non-endothelial nestin + cells encompass early cells of the osteoblast lineage a–c. Nes- GFP; Col2-cre; R26R Tomato femur sections stained for CD31 and nuclei. E12.5, x200 confocal (a) (right panel: CD31 single-color) and E13.5, x200 (b), x630 confocal of dotted area (c). Arrows: CD31 − Nes + Tomato + cells, allowheads: CD31 + Nes + Tomato − cells. d. Nes- GFP; Col2-creER; R26R Tomato E13.5 femur sections stained for CD31 and nuclei. Pregnant mice received 2mg tamoxifen at E12.5. x200 confocal. e, f. Nes- GFP; Osx-creER; R26R Tomato E13.5 femur sections stained for CD31 and nuclei. Pregnant mice received 2mg tamoxifen at E12.5. x200 (e), x630 confocal of the osteogenic perichondrium (f) (lower panel: CD31 single-color). Arrows: CD31 − Nes + Tomato + cells, arrowheads: perivascular Tomato + cells. Green: EGFP, red: tdTomato, blue: Alexa633, gray: DAPI. Scale bars: 50μm (a, b, d, e), 20μm (c, f). g. Flow cytometry analysis of E13.5 Nes- GFP; Osx-creER; R26R Tomato limb cells (tamoxifen at E12.5) stained for CD45. CD45 − fraction gated for Tomato + , blue line: Nes- GFP − Tomato + limb cells (n=3, data represented as mean ± S.D.). h. Nes- GFP; Osx-creER; R26R Tomato E16.5 femur sections (tamoxifen at E13.5) stained for nuclei. Arrowheads: Tomato + cells disappearing from the osteogenic perichondrium. x100. Green: EGFP, red: tdTomato, blue: DAPI. i, j. Nes- GFP; Osx-creER; R26R Tomato E17.5 femur sections (tamoxifen at E16.5) stained for CD31 and nuclei. x100 (i), x630 confocal of dotted area (j). Arrows: CD31 − Nes + Tomato + cells on the innermost layer of the osteogenic perichondrium. Scale bars: 200μm (h, i), 50μm (j). k–m. Nes- GFP; Col2-creER; R26R Tomato P0 femur sections (tamoxifen at E13.5) stained for CD31 and nuclei. x100 (k), x630 confocal of dotted area (l: perichondrium, m: primary spongiosa). Arrows: CD31 − Nes + Tomato + cells. Green: EGFP, red: tdTomato, blue: Alexa633, gray: DAPI. Scale bars: 200μm (k), 20μm (l, m). o–q. E13.5 femur sections stained for CD31 and nuclei. Control (o), Ihh −/ − (p) and Runx2 −/ − (q), x200 confocal. Green: EGFP, red: Alexa546, gray: DAPI. Scale bars: 50μm. r. Quantification of CD31 + Nes + cells and CD31 − Nes + cells in perichondrium. Control (while bars) and Ihh −/ − (black bars), n=4 per group, *p

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

    Nes-creER preferentially targets nestin + endothelial cells in developing bone marrow a. Pregnant mice received 2mg tamoxifen at indicated points (E11.5, 13,5 or 16.5), and Nes-creER; R26R Tomato mice were chased until indicated postnatal days (P0, P7 or P21). Distal femur sections, x40. Red: tdTomato. Scale bars: 500μm. b. Nes-creER; R26R Tomato mice received 0.1mg tamoxifen at P3 and were chased for 1 month (left panels) and 6 months (right panels). Upper panels: distal femur, metaphysis, x40, scale bars: 500μm, lower panels: diaphysis, x100, scale bars: 200μm. Red: tdTomato. c. Flow cytometry analysis of epiphyseal/metaphyseal cells from Nes-creER; R26R Tomato mice received tamoxifen at P3 and chased for indicated periods. Cells were stained for CD45 and CD31. Upper panel: CD45 − fraction after 2 weeks of chase gated for Tomato + , blue line: Tomato + cells, unstained control. Lower panel: Mice were chased for 2 days (n=4), 1 week (n=6), 2 weeks (n=2) and 4 weeks (n=4). The percentage of CD31 + cells within CD45 − Tomato + fraction is shown. d. Nes-creER; R26R Tomato (tamoxifen at P3) femur sections. Left panels: diaphysis after 1 month of chase stained for CD31 and nuclei. Upper left: x200 confocal, arrows: Tomato + osteocytes in cortical bone; Lower left:, x630 confocal of boxed area from upper left, rotated perpendicularly, bone marrow sinusoid. Red: tdTomato, blue: Alexa633, gray: DAPI. Upper right: growth plate cartilage after 2 months of chase. Note Tomato + columnar chondrocytes. x400 confocal, Red: tdTomato, gray: DAPI. Lower right panel: diaphysis after 4 months of chase stained for lipid. x200 confocal, Red: tdTomato, blue: LipidTOX Deep Red. Scale bars: 50μm. e. Flow cytometry analysis of epiphyseal/metaphyseal cells from Col1(2.3 kb)- GFP; Nes-creER; R26R Tomato mice received tamoxifen at P3 and chased for indicated periods. Cells were stained for CD45. Upper panel: CD45 − fraction after 2 days of chase gated for Tomato + , blue line: Tomato + cells, unstained control. Lower panel: Mice were chased for 2 days (n=4), 1 week (n=5), 2 weeks (n=2), 3 weeks (n=4) and 4 weeks (n=4). The percentage of Col1(2.3 kb)- GFP + cells within CD45 − Tomato + fraction is shown. f, g. Cxcl12- GFP; Nes-creER; R26R Tomato mice received tamoxifen at P3 and chased for 1 week. Flow cytometry analysis of epiphyseal/metaphyseal cells (f); CD45 − fraction gated for Tomato + (n=6, blue line: Cxcl12- GFP − Tomato + cells), sections of femur diaphysis bone marrow stained for CD31 and nuclei (g), x630 confocal. Green: EGFP, red: tdTomato, blue: Alexa633, gray: DAPI. Scale bars: 50μm. All data represented as mean ± S.D.
    Figure Legend Snippet: Nes-creER preferentially targets nestin + endothelial cells in developing bone marrow a. Pregnant mice received 2mg tamoxifen at indicated points (E11.5, 13,5 or 16.5), and Nes-creER; R26R Tomato mice were chased until indicated postnatal days (P0, P7 or P21). Distal femur sections, x40. Red: tdTomato. Scale bars: 500μm. b. Nes-creER; R26R Tomato mice received 0.1mg tamoxifen at P3 and were chased for 1 month (left panels) and 6 months (right panels). Upper panels: distal femur, metaphysis, x40, scale bars: 500μm, lower panels: diaphysis, x100, scale bars: 200μm. Red: tdTomato. c. Flow cytometry analysis of epiphyseal/metaphyseal cells from Nes-creER; R26R Tomato mice received tamoxifen at P3 and chased for indicated periods. Cells were stained for CD45 and CD31. Upper panel: CD45 − fraction after 2 weeks of chase gated for Tomato + , blue line: Tomato + cells, unstained control. Lower panel: Mice were chased for 2 days (n=4), 1 week (n=6), 2 weeks (n=2) and 4 weeks (n=4). The percentage of CD31 + cells within CD45 − Tomato + fraction is shown. d. Nes-creER; R26R Tomato (tamoxifen at P3) femur sections. Left panels: diaphysis after 1 month of chase stained for CD31 and nuclei. Upper left: x200 confocal, arrows: Tomato + osteocytes in cortical bone; Lower left:, x630 confocal of boxed area from upper left, rotated perpendicularly, bone marrow sinusoid. Red: tdTomato, blue: Alexa633, gray: DAPI. Upper right: growth plate cartilage after 2 months of chase. Note Tomato + columnar chondrocytes. x400 confocal, Red: tdTomato, gray: DAPI. Lower right panel: diaphysis after 4 months of chase stained for lipid. x200 confocal, Red: tdTomato, blue: LipidTOX Deep Red. Scale bars: 50μm. e. Flow cytometry analysis of epiphyseal/metaphyseal cells from Col1(2.3 kb)- GFP; Nes-creER; R26R Tomato mice received tamoxifen at P3 and chased for indicated periods. Cells were stained for CD45. Upper panel: CD45 − fraction after 2 days of chase gated for Tomato + , blue line: Tomato + cells, unstained control. Lower panel: Mice were chased for 2 days (n=4), 1 week (n=5), 2 weeks (n=2), 3 weeks (n=4) and 4 weeks (n=4). The percentage of Col1(2.3 kb)- GFP + cells within CD45 − Tomato + fraction is shown. f, g. Cxcl12- GFP; Nes-creER; R26R Tomato mice received tamoxifen at P3 and chased for 1 week. Flow cytometry analysis of epiphyseal/metaphyseal cells (f); CD45 − fraction gated for Tomato + (n=6, blue line: Cxcl12- GFP − Tomato + cells), sections of femur diaphysis bone marrow stained for CD31 and nuclei (g), x630 confocal. Green: EGFP, red: tdTomato, blue: Alexa633, gray: DAPI. Scale bars: 50μm. All data represented as mean ± S.D.

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

    25) Product Images from "αvβ3 Integrins Mediate Flow-Induced NF-κB Activation, Proinflammatory Gene Expression, and Early Atherogenic Inflammation"

    Article Title: αvβ3 Integrins Mediate Flow-Induced NF-κB Activation, Proinflammatory Gene Expression, and Early Atherogenic Inflammation

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2015.05.013

    Reduced inflammation after partial carotid ligation in iEC-αv KO mice. A: After tamoxifen treatment, lung endothelial cells were isolated to analyze αv protein levels by Western blot analysis. B: iEC-Control and iEC-αv KO mice
    Figure Legend Snippet: Reduced inflammation after partial carotid ligation in iEC-αv KO mice. A: After tamoxifen treatment, lung endothelial cells were isolated to analyze αv protein levels by Western blot analysis. B: iEC-Control and iEC-αv KO mice

    Techniques Used: Ligation, Mouse Assay, Isolation, Western Blot

    Endothelial αv deletion limits spontaneous atherosclerosis. iEC-Control and iEC-αv KO mice were treated with tamoxifen, and atherosclerosis was induced by Western diet feeding for 8 weeks. A: Oil Red O staining of the aortic arch was performed
    Figure Legend Snippet: Endothelial αv deletion limits spontaneous atherosclerosis. iEC-Control and iEC-αv KO mice were treated with tamoxifen, and atherosclerosis was induced by Western diet feeding for 8 weeks. A: Oil Red O staining of the aortic arch was performed

    Techniques Used: Mouse Assay, Western Blot, Staining

    26) Product Images from "Dose-Dependent Effects of Sirolimus on mTOR Signaling and Polycystic Kidney Disease"

    Article Title: Dose-Dependent Effects of Sirolimus on mTOR Signaling and Polycystic Kidney Disease

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2011040340

    Study design. (A) iKsp- Pkd1 del mice, treated with tamoxifen to disrupt the Pkd1 gene at days 38–40. At day 45, sirolimus was started for the short-term (approximately 80 days) and long-term (105–110 days) treatment groups. At 80 days,
    Figure Legend Snippet: Study design. (A) iKsp- Pkd1 del mice, treated with tamoxifen to disrupt the Pkd1 gene at days 38–40. At day 45, sirolimus was started for the short-term (approximately 80 days) and long-term (105–110 days) treatment groups. At 80 days,

    Techniques Used: Mouse Assay

    27) Product Images from "Ghrelin produces antidepressant-like effect in the estrogen deficient mice"

    Article Title: Ghrelin produces antidepressant-like effect in the estrogen deficient mice

    Journal: Oncotarget

    doi: 10.18632/oncotarget.19768

    Ghrelin possible involve CREB signaling pathway Figures represent the CREB, pCREB and pCREB/CREB ratios in the frontal cortex ( A – C ) and hippocampus ( E , F and J ). Group conditions are indicated by abbreviations and doses by numbers. HP: hippocampus; FC: frontal cortex; Sham: sham treatment; OV: ovariectomy; G: Ghrelin (1 mg/kg); T: tamoxifen (15 mg/kg). Values are mean ± S.E.M, n = 4, Symbols represent significant post hoc comparisons: Tukey's HSD, ** P
    Figure Legend Snippet: Ghrelin possible involve CREB signaling pathway Figures represent the CREB, pCREB and pCREB/CREB ratios in the frontal cortex ( A – C ) and hippocampus ( E , F and J ). Group conditions are indicated by abbreviations and doses by numbers. HP: hippocampus; FC: frontal cortex; Sham: sham treatment; OV: ovariectomy; G: Ghrelin (1 mg/kg); T: tamoxifen (15 mg/kg). Values are mean ± S.E.M, n = 4, Symbols represent significant post hoc comparisons: Tukey's HSD, ** P

    Techniques Used:

    Ghrelin increases the c-Fos-positive cells in hippocampus ( A ) Representative sections through the dentate gyrus (inset: greater magnification near the tip of the dentate), showing c-Fos stained cells (dense brown nuclear staining) and counterstaining with neutral red. ( B ) The number of c-Fos positive cells in the dentate gyrus of the hippocampus. ( C ) The number of c-Fos positive cells in the subregions of the frontal cortex: Cg1, IL, and PrL. Group conditions are indicated by the following letters and abbreviations. HP: hippocampus; FC: frontal cortex; Sham: sham treatment; OV: ovariectomy; G: Ghrelin (1 mg/kg); T: tamoxifen (15 mg/kg). Values are mean ± S.E.M, n = 3–11, Symbols represent significant post hoc comparisons: Tukey's HSD, * P
    Figure Legend Snippet: Ghrelin increases the c-Fos-positive cells in hippocampus ( A ) Representative sections through the dentate gyrus (inset: greater magnification near the tip of the dentate), showing c-Fos stained cells (dense brown nuclear staining) and counterstaining with neutral red. ( B ) The number of c-Fos positive cells in the dentate gyrus of the hippocampus. ( C ) The number of c-Fos positive cells in the subregions of the frontal cortex: Cg1, IL, and PrL. Group conditions are indicated by the following letters and abbreviations. HP: hippocampus; FC: frontal cortex; Sham: sham treatment; OV: ovariectomy; G: Ghrelin (1 mg/kg); T: tamoxifen (15 mg/kg). Values are mean ± S.E.M, n = 3–11, Symbols represent significant post hoc comparisons: Tukey's HSD, * P

    Techniques Used: Staining

    Ovariectomy decreases the c-Fos-positive cells in hippocampus Representative sections through the hippocampus (such as: CA1, CA2, CA3, CA4), showing c-Fos stained cells (dense brown nuclear staining) and counterstaining with neutral red. Group conditions are indicated by the following letters and abbreviations. HP: hippocampus; CA1, field CA1 of hippocampus; CA2, field CA2 of hippocampus; CA3, field CA3 of hippocampus; CA4, field CA4 of hippocampus; Sham: sham treatment; OV: ovariectomy; G: Ghrelin (1 mg/kg); T: tamoxifen (15 mg/kg). Values are mean ± S.E.M, n = 5–11, Symbols represent significant post hoc comparisons: Tukey's HSD, * P
    Figure Legend Snippet: Ovariectomy decreases the c-Fos-positive cells in hippocampus Representative sections through the hippocampus (such as: CA1, CA2, CA3, CA4), showing c-Fos stained cells (dense brown nuclear staining) and counterstaining with neutral red. Group conditions are indicated by the following letters and abbreviations. HP: hippocampus; CA1, field CA1 of hippocampus; CA2, field CA2 of hippocampus; CA3, field CA3 of hippocampus; CA4, field CA4 of hippocampus; Sham: sham treatment; OV: ovariectomy; G: Ghrelin (1 mg/kg); T: tamoxifen (15 mg/kg). Values are mean ± S.E.M, n = 5–11, Symbols represent significant post hoc comparisons: Tukey's HSD, * P

    Techniques Used: Staining

    Ghrelin increased BrdU expression in the dendate gyrus of the hippocampus The arrows show BrdU stained cells. Group conditions are indicated by the following letters and abbreviations: sham (sham treatment), OV (ovariectomy), G (Ghrelin, 1 mg/kg) and T (tamoxifen, 15 mg/kg).
    Figure Legend Snippet: Ghrelin increased BrdU expression in the dendate gyrus of the hippocampus The arrows show BrdU stained cells. Group conditions are indicated by the following letters and abbreviations: sham (sham treatment), OV (ovariectomy), G (Ghrelin, 1 mg/kg) and T (tamoxifen, 15 mg/kg).

    Techniques Used: Expressing, Staining

    Ghrelin possible involve BDNF signaling pathway Figures represent the BDNF levels (normalized to β-actin) in the frontal cortex ( A ) and hippocampus ( B ). Group conditions are indicated by abbreviations and doses by numbers. HP: hippocampus; FC: frontal cortex; Sham: sham treatment; OV: ovariectomy; G: ghrelin (1 mg/kg); T: tamoxifen (15 mg/kg). Values are mean ± S.E.M, n = 4, Symbols represent significant post hoc comparisons: Tukey's HSD, * P
    Figure Legend Snippet: Ghrelin possible involve BDNF signaling pathway Figures represent the BDNF levels (normalized to β-actin) in the frontal cortex ( A ) and hippocampus ( B ). Group conditions are indicated by abbreviations and doses by numbers. HP: hippocampus; FC: frontal cortex; Sham: sham treatment; OV: ovariectomy; G: ghrelin (1 mg/kg); T: tamoxifen (15 mg/kg). Values are mean ± S.E.M, n = 4, Symbols represent significant post hoc comparisons: Tukey's HSD, * P

    Techniques Used:

    Ghrelin alleviate depression behavior ( A ) Locomotor behavior (number of line crosses) in the open field. ( B ) Rearing behavior (frequency) in the open field. Group conditions are indicated by abbreviations, and doses by numbers. ( C ) Group conditions are indicated by abbreviations, and doses by numbers. Sham: sham treatment; OV: ovariectomy; G: Ghrelin (1 mg/kg); T: tamoxifen (15 mg/kg). Values are mean ± S.E.M, n = 5–8, Symbols represent significant post hoc comparisons: Tukey's HSD, ** P
    Figure Legend Snippet: Ghrelin alleviate depression behavior ( A ) Locomotor behavior (number of line crosses) in the open field. ( B ) Rearing behavior (frequency) in the open field. Group conditions are indicated by abbreviations, and doses by numbers. ( C ) Group conditions are indicated by abbreviations, and doses by numbers. Sham: sham treatment; OV: ovariectomy; G: Ghrelin (1 mg/kg); T: tamoxifen (15 mg/kg). Values are mean ± S.E.M, n = 5–8, Symbols represent significant post hoc comparisons: Tukey's HSD, ** P

    Techniques Used:

    28) Product Images from "Multi-color lineage tracing reveals clonal dynamics of squamous carcinoma evolution from initiation to metastasis"

    Article Title: Multi-color lineage tracing reveals clonal dynamics of squamous carcinoma evolution from initiation to metastasis

    Journal: Nature cell biology

    doi: 10.1038/s41556-018-0109-0

    Tamoxifen-induced Confetti labeling of the skin. (A and B) Back skin sections of K5CreER-Confetti mice (5 mice), treated with 4 doses of tamoxifen to activate Confetti recombination. Sections taken 10 days after final dose of tamoxifen, at 10× (A) and 40× (B) magnification. (C) Proportion of labeled skin cells in back skin expressing each Confetti fluorophore, 10 days after final dose of tamoxifen (n=3 mice, 44 panels). See Supplementary Table 3 for statistics source data. (D) Schematic of tumorigenesis strategy. K5CreER-Confetti mice are treated with 4 doses of tamoxifen to activate Confetti labeling, and then treated with the carcinogen DMBA 10 days after final tamoxifen dose, followed by biweekly treatments with tumor promoter TPA. Dozens of benign papillomas emerge beginning at 6-8 weeks, a subset of which progress to carcinomas. Carcinomas are surgically resected when they reach 1cm in diameter, and mice go on to develop metastatic disease.
    Figure Legend Snippet: Tamoxifen-induced Confetti labeling of the skin. (A and B) Back skin sections of K5CreER-Confetti mice (5 mice), treated with 4 doses of tamoxifen to activate Confetti recombination. Sections taken 10 days after final dose of tamoxifen, at 10× (A) and 40× (B) magnification. (C) Proportion of labeled skin cells in back skin expressing each Confetti fluorophore, 10 days after final dose of tamoxifen (n=3 mice, 44 panels). See Supplementary Table 3 for statistics source data. (D) Schematic of tumorigenesis strategy. K5CreER-Confetti mice are treated with 4 doses of tamoxifen to activate Confetti labeling, and then treated with the carcinogen DMBA 10 days after final tamoxifen dose, followed by biweekly treatments with tumor promoter TPA. Dozens of benign papillomas emerge beginning at 6-8 weeks, a subset of which progress to carcinomas. Carcinomas are surgically resected when they reach 1cm in diameter, and mice go on to develop metastatic disease.

    Techniques Used: Labeling, Mouse Assay, Expressing

    Tumor evolution following Confetti labeling at 8 weeks. (A) Schematic of experimental design. DMBA/TPA-induced papillomas were allowed to grow for 8 weeks in K5CreER-Confetti mice. Confetti labeling was subsequently activated 8 weeks after DMBA treatment with 2 doses of tamoxifen, and TPA treatment was continued. (B) Cross-section of a papilloma labeled at 8 weeks post-DMBA, 3 days after final dose of tamoxifen, exhibiting Confetti labeling (3-day labeling verification based on 3 tumors). (C, D) Whole papillomas labeled at 8 weeks post-DMBA (25 papillomas), viewed from with side with a fluorescent dissecting microscope. Images taken 8 weeks (D) and 27 weeks (C) after tamoxifen. Green and red lobes are visible in (C) and right-hand tumor in (D), blue lobe and small red lobe are visible in left-hand tumor in (D). (E) Cross-section of right-hand tumor in (D), a papilloma labeled at 8 weeks post-DMBA. Image taken 8 weeks after tamoxifen. Nuclei are marked with DAPI. (F and G) High magnification images of tumor shown in (E), highlighting boundaries between colored lobes where limited intermixing is seen. Nuclei are marked with DAPI. (H and I) Cross-sections of carcinomas from mice in which Confetti labeling was activated 8 weeks post-DMBA, each exhibiting only a single color (13 carcinomas). Nuclei are marked with DAPI. (J) Whole carcinoma from a mouse treated with tamoxifen at 8 weeks (13 carcinomas), viewed from the top (left) and side (right). Streaks, indicated by arrows, are visible but confined to the periphery.
    Figure Legend Snippet: Tumor evolution following Confetti labeling at 8 weeks. (A) Schematic of experimental design. DMBA/TPA-induced papillomas were allowed to grow for 8 weeks in K5CreER-Confetti mice. Confetti labeling was subsequently activated 8 weeks after DMBA treatment with 2 doses of tamoxifen, and TPA treatment was continued. (B) Cross-section of a papilloma labeled at 8 weeks post-DMBA, 3 days after final dose of tamoxifen, exhibiting Confetti labeling (3-day labeling verification based on 3 tumors). (C, D) Whole papillomas labeled at 8 weeks post-DMBA (25 papillomas), viewed from with side with a fluorescent dissecting microscope. Images taken 8 weeks (D) and 27 weeks (C) after tamoxifen. Green and red lobes are visible in (C) and right-hand tumor in (D), blue lobe and small red lobe are visible in left-hand tumor in (D). (E) Cross-section of right-hand tumor in (D), a papilloma labeled at 8 weeks post-DMBA. Image taken 8 weeks after tamoxifen. Nuclei are marked with DAPI. (F and G) High magnification images of tumor shown in (E), highlighting boundaries between colored lobes where limited intermixing is seen. Nuclei are marked with DAPI. (H and I) Cross-sections of carcinomas from mice in which Confetti labeling was activated 8 weeks post-DMBA, each exhibiting only a single color (13 carcinomas). Nuclei are marked with DAPI. (J) Whole carcinoma from a mouse treated with tamoxifen at 8 weeks (13 carcinomas), viewed from the top (left) and side (right). Streaks, indicated by arrows, are visible but confined to the periphery.

    Techniques Used: Labeling, Mouse Assay, Microscopy

    Carcinoma evolution following Confetti labeling at 24 weeks. (A) Schematic of experimental design. DMBA/TPA-induced papillomas were allowed to grow for 24 weeks in K5CreER-Confetti mice. Confetti labeling was subsequently activated with 2 doses of tamoxifen. (B) Cross-section of a papilloma labeled at 24 weeks post-DMBA, 3 days after final dose of tamoxifen, exhibiting Confetti labeling (3-day labeling verification based on 2 tumors). (C) Relationship between labeling pattern observed in harvested carcinomas and the time elapsed between tamoxifen labeling and harvest. Carcinomas harvested close to time of labeling were frequently multi-color ( n = 5), carcinomas harvested at an intermediate time point were frequently speckled ( n = 7), and carcinomas harvested long after labeling were typically single colored ( n = 9). Asterisk (*) indicates P
    Figure Legend Snippet: Carcinoma evolution following Confetti labeling at 24 weeks. (A) Schematic of experimental design. DMBA/TPA-induced papillomas were allowed to grow for 24 weeks in K5CreER-Confetti mice. Confetti labeling was subsequently activated with 2 doses of tamoxifen. (B) Cross-section of a papilloma labeled at 24 weeks post-DMBA, 3 days after final dose of tamoxifen, exhibiting Confetti labeling (3-day labeling verification based on 2 tumors). (C) Relationship between labeling pattern observed in harvested carcinomas and the time elapsed between tamoxifen labeling and harvest. Carcinomas harvested close to time of labeling were frequently multi-color ( n = 5), carcinomas harvested at an intermediate time point were frequently speckled ( n = 7), and carcinomas harvested long after labeling were typically single colored ( n = 9). Asterisk (*) indicates P

    Techniques Used: Labeling, Mouse Assay

    29) Product Images from "Interaction of HIF1α and β-catenin inhibits matrix metalloproteinase 13 expression and prevents cartilage damage in mice"

    Article Title: Interaction of HIF1α and β-catenin inhibits matrix metalloproteinase 13 expression and prevents cartilage damage in mice

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1514854113

    β-Galactosidase immunostaining in Col2-Cre ERT ; R26R- LacZ and R26R- LacZ mice with injection of tamoxifen ( n = 3).
    Figure Legend Snippet: β-Galactosidase immunostaining in Col2-Cre ERT ; R26R- LacZ and R26R- LacZ mice with injection of tamoxifen ( n = 3).

    Techniques Used: Immunostaining, Mouse Assay, Injection

    30) Product Images from "Protein Phosphatase 2A B55 and A Regulatory Subunits Interact with Nitrate Reductase and Are Essential for Nitrate Reductase Activation 1Protein Phosphatase 2A B55 and A Regulatory Subunits Interact with Nitrate Reductase and Are Essential for Nitrate Reductase Activation 1 [W]Protein Phosphatase 2A B55 and A Regulatory Subunits Interact with Nitrate Reductase and Are Essential for Nitrate Reductase Activation 1 [W] [OA]"

    Article Title: Protein Phosphatase 2A B55 and A Regulatory Subunits Interact with Nitrate Reductase and Are Essential for Nitrate Reductase Activation 1Protein Phosphatase 2A B55 and A Regulatory Subunits Interact with Nitrate Reductase and Are Essential for Nitrate Reductase Activation 1 [W]Protein Phosphatase 2A B55 and A Regulatory Subunits Interact with Nitrate Reductase and Are Essential for Nitrate Reductase Activation 1 [W] [OA]

    Journal: Plant Physiology

    doi: 10.1104/pp.111.172734

    The scaffolding A subunits of PP2A are required for light activation of NR. A, Relative transcript levels for the PP2AA subunit in tamoxifen-treated wild-type and amiRNA-1 plants, and mock-treated plants. Expression levels were tested with RT-PCR using
    Figure Legend Snippet: The scaffolding A subunits of PP2A are required for light activation of NR. A, Relative transcript levels for the PP2AA subunit in tamoxifen-treated wild-type and amiRNA-1 plants, and mock-treated plants. Expression levels were tested with RT-PCR using

    Techniques Used: Scaffolding, Activation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction

    31) Product Images from "NLRP3 inflammasome activation in hepatic stellate cells induces murine liver fibrosis"

    Article Title: NLRP3 inflammasome activation in hepatic stellate cells induces murine liver fibrosis

    Journal: Hepatology (Baltimore, Md.)

    doi: 10.1002/hep.30252

    Effect of NLRP3 inflammasome activation in hepatic stellate cells fibrotic phenotype After LPS/ATP stimulation, HSC from WT but not Nlrp3 −/− mice showed an increase in collagen 1 and αSMA expression along with a change in the shape (A). Flow cytometry analysis showed that WT HSC displayed increased vimentin levels after LPS/ATP stimulation, an effect not present in Nlrp3 −/− HSC (B). mRNA levels of Ctgf , aSma , Tgfb , Vim , and Coll1a1 were found to be increased after LPS, and further enhanced after subsequent ATP stimulation (C). HSC isolated from Nlrp3 L351P/+ CreT showed a pronounced change in shape along with increased collagen 1 and αSMA expression (D), augmented vimentin expression measured by FACS (E) and elevated mRNA levels of aSma , Coll1a1 , Vim , Ctgf and Tgfb (F) after NLRP3 inflammasome overactivation was induced by 4-OH-tamoxifen, effects that were intensified after the addition of LPS. (6 replicates were included for all measured values, * = p
    Figure Legend Snippet: Effect of NLRP3 inflammasome activation in hepatic stellate cells fibrotic phenotype After LPS/ATP stimulation, HSC from WT but not Nlrp3 −/− mice showed an increase in collagen 1 and αSMA expression along with a change in the shape (A). Flow cytometry analysis showed that WT HSC displayed increased vimentin levels after LPS/ATP stimulation, an effect not present in Nlrp3 −/− HSC (B). mRNA levels of Ctgf , aSma , Tgfb , Vim , and Coll1a1 were found to be increased after LPS, and further enhanced after subsequent ATP stimulation (C). HSC isolated from Nlrp3 L351P/+ CreT showed a pronounced change in shape along with increased collagen 1 and αSMA expression (D), augmented vimentin expression measured by FACS (E) and elevated mRNA levels of aSma , Coll1a1 , Vim , Ctgf and Tgfb (F) after NLRP3 inflammasome overactivation was induced by 4-OH-tamoxifen, effects that were intensified after the addition of LPS. (6 replicates were included for all measured values, * = p

    Techniques Used: Activation Assay, Mouse Assay, Expressing, Flow Cytometry, Cytometry, Isolation, FACS

    32) Product Images from "Effects of SERM (selective estrogen receptor modulator) treatment on growth and proliferation in the rat uterus"

    Article Title: Effects of SERM (selective estrogen receptor modulator) treatment on growth and proliferation in the rat uterus

    Journal: Reproductive biology and endocrinology : RB & E

    doi: 10.1186/1477-7827-1-40

    The estrogen receptor α (ERα) mRNA (amol/μg DNA), estrogen receptor β (ERβ) mRNA (amol/μg DNA), and the ratio of ERα mRNA and ERβ mRNA, in the ovariectomized (OVX) controls and in the rats treated with estradiol (E 2 ), tamoxifen (TAM), raloxifene (RAL) or ICI182780 (ICI). Box and whisker plots representing the median value with 50% of all data falling within the box. The whiskers extend to the 5 th and 95 th percentiles. Boxes with different letter designations are significantly different (p
    Figure Legend Snippet: The estrogen receptor α (ERα) mRNA (amol/μg DNA), estrogen receptor β (ERβ) mRNA (amol/μg DNA), and the ratio of ERα mRNA and ERβ mRNA, in the ovariectomized (OVX) controls and in the rats treated with estradiol (E 2 ), tamoxifen (TAM), raloxifene (RAL) or ICI182780 (ICI). Box and whisker plots representing the median value with 50% of all data falling within the box. The whiskers extend to the 5 th and 95 th percentiles. Boxes with different letter designations are significantly different (p

    Techniques Used: Whisker Assay

    The uterine wet weight (g), luminal epithelium (LE) height (μm), growth hormone receptor (GH-R) mRNA (amol/μg DNA), and insulin-like growth factor-I (IGF-I) mRNA (amol/μg DNA), in the ovariectomized (OVX) controls and in the rats treated with estradiol (E 2 ), tamoxifen (TAM), raloxifene (RAL) or ICI182780 (ICI). Box and whisker plots representing the median value with 50% of all data falling within the box. The whiskers extend to the 5 th and 95 th percentiles. Boxes with different letter designations are significantly different (p
    Figure Legend Snippet: The uterine wet weight (g), luminal epithelium (LE) height (μm), growth hormone receptor (GH-R) mRNA (amol/μg DNA), and insulin-like growth factor-I (IGF-I) mRNA (amol/μg DNA), in the ovariectomized (OVX) controls and in the rats treated with estradiol (E 2 ), tamoxifen (TAM), raloxifene (RAL) or ICI182780 (ICI). Box and whisker plots representing the median value with 50% of all data falling within the box. The whiskers extend to the 5 th and 95 th percentiles. Boxes with different letter designations are significantly different (p

    Techniques Used: Whisker Assay

    33) Product Images from "Protein Disulfide Isomerase-associated 6 is an ATF6-inducible ER Stress Response Protein that Protects Cardiac Myocytes from Ischemia/Reperfusion-mediated Cell Death"

    Article Title: Protein Disulfide Isomerase-associated 6 is an ATF6-inducible ER Stress Response Protein that Protects Cardiac Myocytes from Ischemia/Reperfusion-mediated Cell Death

    Journal: Journal of molecular and cellular cardiology

    doi: 10.1016/j.yjmcc.2012.05.005

    PDIA6 expression in ATF6 TG mouse hearts and ATF6 binding to the PDIA6 promoter in mouse hearts, in vivo Panel A - NTG and ATF6 TG mice were treated with or without tamoxifen for 5 days, after which hearts were extracted and analyzed for PDIA6 and GAPDH mRNA by qRT-PCR. Values are mean PDIA6/GAPDH mRNA ± SE, n=3 mice per treatment. Panel B - Quantitative ChIP analysis was carried out to determine the ability of ATF6 to bind to the PDIA6 and hemoxygenase-1 (HO-1) promoters in the mouse heart, in vivo . Chromatin was isolated from NTG and ATF6 TG mouse hearts, and then subjected to FLAG IP. Isolated chromatin was then examined by quantitative PCR using primers targeted to the mouse PDIA6 and mouse HO-1 promoters. Shown is the quantitation of the PCR products obtained, n=3 mice per treatment.
    Figure Legend Snippet: PDIA6 expression in ATF6 TG mouse hearts and ATF6 binding to the PDIA6 promoter in mouse hearts, in vivo Panel A - NTG and ATF6 TG mice were treated with or without tamoxifen for 5 days, after which hearts were extracted and analyzed for PDIA6 and GAPDH mRNA by qRT-PCR. Values are mean PDIA6/GAPDH mRNA ± SE, n=3 mice per treatment. Panel B - Quantitative ChIP analysis was carried out to determine the ability of ATF6 to bind to the PDIA6 and hemoxygenase-1 (HO-1) promoters in the mouse heart, in vivo . Chromatin was isolated from NTG and ATF6 TG mouse hearts, and then subjected to FLAG IP. Isolated chromatin was then examined by quantitative PCR using primers targeted to the mouse PDIA6 and mouse HO-1 promoters. Shown is the quantitation of the PCR products obtained, n=3 mice per treatment.

    Techniques Used: Expressing, Binding Assay, In Vivo, Mouse Assay, Quantitative RT-PCR, Chromatin Immunoprecipitation, Isolation, Real-time Polymerase Chain Reaction, Quantitation Assay, Polymerase Chain Reaction

    34) Product Images from "Ablation of huntingtin in adult neurons is nondeleterious but its depletion in young mice causes acute pancreatitis"

    Article Title: Ablation of huntingtin in adult neurons is nondeleterious but its depletion in young mice causes acute pancreatitis

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1524575113

    Rescued effect of transgenic tHTT on Htt KO-induced pancreatitis. ( A ) tHTT construct used for generation of the tHTT transgenic mice. ( B ) Western blotting showing that tHTT is expressed in the mouse brain cortex. Wild-type mice cortex was used as control. ( C ) PCR genotyping verified the presence of transgenic tHTT in heterozygous ( tHTT /Ctl) and homozygous ( tHTT /KO) ubiquitous KO mice that also carry CAG-CreER. Ctl, control. ( D ) Survival rate of tHTT /KO mouse and ubiquitous KO mice showing that tHTT can prevent early death. ( E ) Body weight was tested at the fifth month after tamoxifen injection in the tHTT /KO and control ( tHTT /Ctl) mice (paired two-tailed t test; n.s. represents no significant difference, n = 8, tHTT /KO, male = 5, female = 3; control, male = 4, female = 4). ( F ) Western blotting showing that tHTT is expressed in the ubiquitous KO mouse pancreas; heterozygous KO mouse pancreas was used as control. Note tHTT is smaller than full-length Htt (Htt). Three mouse pancreases of each genotype were examined. ( G ) Western blotting of the CELA3B, caspase-3, P62, RIPK3, and LC3I/II protein in the 2-mo-old tHTT /KO and control mouse pancreas.
    Figure Legend Snippet: Rescued effect of transgenic tHTT on Htt KO-induced pancreatitis. ( A ) tHTT construct used for generation of the tHTT transgenic mice. ( B ) Western blotting showing that tHTT is expressed in the mouse brain cortex. Wild-type mice cortex was used as control. ( C ) PCR genotyping verified the presence of transgenic tHTT in heterozygous ( tHTT /Ctl) and homozygous ( tHTT /KO) ubiquitous KO mice that also carry CAG-CreER. Ctl, control. ( D ) Survival rate of tHTT /KO mouse and ubiquitous KO mice showing that tHTT can prevent early death. ( E ) Body weight was tested at the fifth month after tamoxifen injection in the tHTT /KO and control ( tHTT /Ctl) mice (paired two-tailed t test; n.s. represents no significant difference, n = 8, tHTT /KO, male = 5, female = 3; control, male = 4, female = 4). ( F ) Western blotting showing that tHTT is expressed in the ubiquitous KO mouse pancreas; heterozygous KO mouse pancreas was used as control. Note tHTT is smaller than full-length Htt (Htt). Three mouse pancreases of each genotype were examined. ( G ) Western blotting of the CELA3B, caspase-3, P62, RIPK3, and LC3I/II protein in the 2-mo-old tHTT /KO and control mouse pancreas.

    Techniques Used: Transgenic Assay, Construct, Mouse Assay, Western Blot, Polymerase Chain Reaction, CTL Assay, Injection, Two Tailed Test

    The expression of autophagy markers in the brain and peripheral tissues of adult ubiquitous Htt KO mice. ( A – C ) Representative immunoblots ( A ) and quantification of ( B ) Htt, P62, and ( C ) LC3I/II ratio in ubiquitous KO and control mice ( n = 3 independent experiments). ( D and E ) Western blotting of the spleen ( D ) and liver ( E ) in control and ubiquitous KO mice. The blots were probed by antibodies to cleaved caspase3, P62, LC3I/II, NF-κB, and FAT10 after tamoxifen injection for 3 d. Alpha-tubulin was probed as a loading control. Animal ID numbers are indicated. Ctl: heterozygous floxed Htt /CAG-CreER mice injected with tamoxifen; KO: homozygous floxed Htt /CAG-CreER mice injected with tamoxifen.
    Figure Legend Snippet: The expression of autophagy markers in the brain and peripheral tissues of adult ubiquitous Htt KO mice. ( A – C ) Representative immunoblots ( A ) and quantification of ( B ) Htt, P62, and ( C ) LC3I/II ratio in ubiquitous KO and control mice ( n = 3 independent experiments). ( D and E ) Western blotting of the spleen ( D ) and liver ( E ) in control and ubiquitous KO mice. The blots were probed by antibodies to cleaved caspase3, P62, LC3I/II, NF-κB, and FAT10 after tamoxifen injection for 3 d. Alpha-tubulin was probed as a loading control. Animal ID numbers are indicated. Ctl: heterozygous floxed Htt /CAG-CreER mice injected with tamoxifen; KO: homozygous floxed Htt /CAG-CreER mice injected with tamoxifen.

    Techniques Used: Expressing, Mouse Assay, Western Blot, Injection, CTL Assay

    Neuronal Htt KO mice brain. ( A ) Photographs of H E-stained coronal sections of the control and neuronal Htt KO mouse brain at the 90th day after tamoxifen injection. (Scale bar, 100 μm.) ( B ) Immunostaining of β-tubulin, cleaved caspase3, GFAP, and LC3I/II in the brains of neuronal Htt KO and control mice at the 90th day after tamoxifen induction. The relative numbers of NeuN- or GFAP-positive cells are shown below the images. TAM: tamoxifen. [Scale bar, 50 μm ( Left ), 200 μm ( Right ).] Ctl: heterozygous floxed Htt /Nestin-Cre mice injected with tamoxifen; oil: homozygous floxed Htt /Nestin-Cre mice injected with corn oil; KO: homozygous floxed Htt /Nestin-Cre mice injected with tamoxifen.
    Figure Legend Snippet: Neuronal Htt KO mice brain. ( A ) Photographs of H E-stained coronal sections of the control and neuronal Htt KO mouse brain at the 90th day after tamoxifen injection. (Scale bar, 100 μm.) ( B ) Immunostaining of β-tubulin, cleaved caspase3, GFAP, and LC3I/II in the brains of neuronal Htt KO and control mice at the 90th day after tamoxifen induction. The relative numbers of NeuN- or GFAP-positive cells are shown below the images. TAM: tamoxifen. [Scale bar, 50 μm ( Left ), 200 μm ( Right ).] Ctl: heterozygous floxed Htt /Nestin-Cre mice injected with tamoxifen; oil: homozygous floxed Htt /Nestin-Cre mice injected with corn oil; KO: homozygous floxed Htt /Nestin-Cre mice injected with tamoxifen.

    Techniques Used: Mouse Assay, Staining, Injection, Immunostaining, CTL Assay

    Neuronal Htt knockout mice. ( A ) Western blots showing depletion of Htt in the brain of neuronal KO mice at 4 and 8 mo of age. See Fig. 1 for abbreviations key. ( B ) Survival rate of neuronal KO mice compared with the heterozygous control mice. Data were collected from the first day after tamoxifen injection. ( C ) Diagram showing the body weight, rotarod performance, and gripping ability at different months. Mice at different ages (2, 4, and 8 mo) were studied for 7–8 mo after tamoxifen injection (2-mo-old, n = 10; KO, male = 6 and female = 4; control, male = 4 and female = 6; 4-mo-old, n = 10; KO, male = 5 and female = 5; control, male = 5 and female = 5; 8-mo-old, KO, n = 9, male = 4, female = 5; control, n = 11, male = 6, female = 5; two-way ANOVA test, body weight, 2M control vs. 2M KO: P = 0.9945, 4M control vs. 4M KO: P = 0.9862, 8M control vs. 8M KO P = 0.9922; rotatod performance, 2M control vs. 2M KO: P = 0.9565, 4M control vs. 4M KO: P = 0.9921, 8M control vs. 8M KO P = 0.7701; gripping ability, 2M control vs. 2M KO: P = 1.0000, 4M control vs. 4M KO: P = 0.9962, 8M control vs. 8M KO P = 0.9718).
    Figure Legend Snippet: Neuronal Htt knockout mice. ( A ) Western blots showing depletion of Htt in the brain of neuronal KO mice at 4 and 8 mo of age. See Fig. 1 for abbreviations key. ( B ) Survival rate of neuronal KO mice compared with the heterozygous control mice. Data were collected from the first day after tamoxifen injection. ( C ) Diagram showing the body weight, rotarod performance, and gripping ability at different months. Mice at different ages (2, 4, and 8 mo) were studied for 7–8 mo after tamoxifen injection (2-mo-old, n = 10; KO, male = 6 and female = 4; control, male = 4 and female = 6; 4-mo-old, n = 10; KO, male = 5 and female = 5; control, male = 5 and female = 5; 8-mo-old, KO, n = 9, male = 4, female = 5; control, n = 11, male = 6, female = 5; two-way ANOVA test, body weight, 2M control vs. 2M KO: P = 0.9945, 4M control vs. 4M KO: P = 0.9862, 8M control vs. 8M KO P = 0.9922; rotatod performance, 2M control vs. 2M KO: P = 0.9565, 4M control vs. 4M KO: P = 0.9921, 8M control vs. 8M KO P = 0.7701; gripping ability, 2M control vs. 2M KO: P = 1.0000, 4M control vs. 4M KO: P = 0.9962, 8M control vs. 8M KO P = 0.9718).

    Techniques Used: Knock-Out, Mouse Assay, Western Blot, Injection

    Histology of pancreas in ubiquitous Htt KO mice at 2 mo of age. ( A ) Intestine morphology in 2-mo-old ubiquitous KO or control mice injected with corn oil or tamoxifen. (Scale bar, 5 mm.) ( B ) H E staining of the acinar cells and islets in the pancreas in the control and ubiquitous KO mice at 2, 4, and 8 mo of age after tamoxifen injection for 5 d. (Scale bar, 10 μm.) ( C ) Pancreas trypsinogen and pancreas amylase activity were measured after TAM injection 40 h in ubiquitous KO and control mice. Data represent the means of eight animals of control and ubiquitous KO mice at each interval ± SEM (* P
    Figure Legend Snippet: Histology of pancreas in ubiquitous Htt KO mice at 2 mo of age. ( A ) Intestine morphology in 2-mo-old ubiquitous KO or control mice injected with corn oil or tamoxifen. (Scale bar, 5 mm.) ( B ) H E staining of the acinar cells and islets in the pancreas in the control and ubiquitous KO mice at 2, 4, and 8 mo of age after tamoxifen injection for 5 d. (Scale bar, 10 μm.) ( C ) Pancreas trypsinogen and pancreas amylase activity were measured after TAM injection 40 h in ubiquitous KO and control mice. Data represent the means of eight animals of control and ubiquitous KO mice at each interval ± SEM (* P

    Techniques Used: Mouse Assay, Injection, Staining, Activity Assay

    Loss of Htt-mediated age-dependent death in mice. Control is heterozygous floxed Htt /CAG-CreER mice injected with tamoxifen, and KO is homozygous floxed Htt /CAG-CreER mice injected with tamoxifen. ( A ) Diagram depicting inactivation of the Htt gene in adult mouse expressing CreER. The mice were intraperitoneally (i.p.) injected with tamoxifen (TAM) 20 mg/mL for 5 continuous days. Mice were examined for 7–11 mo after tamoxifen injection. ( B ) Western blots showing relative Htt protein levels in the brain of ubiquitous KO and control mice at 2, 4, and 8 mo of age. Ctx, cortex; cere, cerebellum; bs, brainstem; str, striatum; oth, other brain regions. ( C ) Survival rate of ubiquitous KO mouse and control mice when they were injected with tamoxifen at 2, 4, and 8 mo of age. ( D and E ) The rotarod performance ( D ) and body weight ( E ) of ubiquitous KO mice whose Htt was depleted at 4 or 8 mo of age, the mice were then studied for 10–11 mo after tamoxifen injection. Control is age-matched heterozygous KO mice.
    Figure Legend Snippet: Loss of Htt-mediated age-dependent death in mice. Control is heterozygous floxed Htt /CAG-CreER mice injected with tamoxifen, and KO is homozygous floxed Htt /CAG-CreER mice injected with tamoxifen. ( A ) Diagram depicting inactivation of the Htt gene in adult mouse expressing CreER. The mice were intraperitoneally (i.p.) injected with tamoxifen (TAM) 20 mg/mL for 5 continuous days. Mice were examined for 7–11 mo after tamoxifen injection. ( B ) Western blots showing relative Htt protein levels in the brain of ubiquitous KO and control mice at 2, 4, and 8 mo of age. Ctx, cortex; cere, cerebellum; bs, brainstem; str, striatum; oth, other brain regions. ( C ) Survival rate of ubiquitous KO mouse and control mice when they were injected with tamoxifen at 2, 4, and 8 mo of age. ( D and E ) The rotarod performance ( D ) and body weight ( E ) of ubiquitous KO mice whose Htt was depleted at 4 or 8 mo of age, the mice were then studied for 10–11 mo after tamoxifen injection. Control is age-matched heterozygous KO mice.

    Techniques Used: Mouse Assay, Injection, Expressing, Western Blot

    Forebrain Htt knockout does not affect mice survival. Inactivation of Htt in the forebrain is mediated by tamoxifen injection into floxed Htt mice expressing CaMKII promoter-driven CreER. ( A ) Body weights of floxed Htt mice expressing CaMKII-CreER and control mice from birth to adult without tamoxifen injection. * P
    Figure Legend Snippet: Forebrain Htt knockout does not affect mice survival. Inactivation of Htt in the forebrain is mediated by tamoxifen injection into floxed Htt mice expressing CaMKII promoter-driven CreER. ( A ) Body weights of floxed Htt mice expressing CaMKII-CreER and control mice from birth to adult without tamoxifen injection. * P

    Techniques Used: Knock-Out, Mouse Assay, Injection, Expressing

    35) Product Images from "Generation of CD4CreERT2 transgenic mice to study development of peripheral CD4-T-cells"

    Article Title: Generation of CD4CreERT2 transgenic mice to study development of peripheral CD4-T-cells

    Journal: Genesis (New York, N.Y. : 2000)

    doi: 10.1002/dvg.22052

    In-vivo Cre activation The indicated mice were treated in-vivo with tamoxifen (See Methods). EYFP expression was analyzed as a measure of Cre activation and representative FACS plots of the indicated parameters are shown. In ( a ) histogram plots show EYFP expression in un-gated live cells from the spleen of the indicated mice. Tow parameter contour plots show CD4 versus CD8 expression in gated YFP+ and YFP− cells as shown by the arrows. ( b ) Line histograms show EYFP expression in cells harvested from the spleen of the indicated mice and gated for surface expression of the indicated markers. Data are representative of three independent experiments.
    Figure Legend Snippet: In-vivo Cre activation The indicated mice were treated in-vivo with tamoxifen (See Methods). EYFP expression was analyzed as a measure of Cre activation and representative FACS plots of the indicated parameters are shown. In ( a ) histogram plots show EYFP expression in un-gated live cells from the spleen of the indicated mice. Tow parameter contour plots show CD4 versus CD8 expression in gated YFP+ and YFP− cells as shown by the arrows. ( b ) Line histograms show EYFP expression in cells harvested from the spleen of the indicated mice and gated for surface expression of the indicated markers. Data are representative of three independent experiments.

    Techniques Used: In Vivo, Activation Assay, Mouse Assay, Expressing, FACS

    36) Product Images from "Effects of estrogens and antiestrogens on gonadal sex differentiation and embryonic development in the domestic fowl (Gallus gallus domesticus)"

    Article Title: Effects of estrogens and antiestrogens on gonadal sex differentiation and embryonic development in the domestic fowl (Gallus gallus domesticus)

    Journal: PeerJ

    doi: 10.7717/peerj.5094

    Effects of in ovo exposure to tamoxifen and 17 α -ethinylestradiol on mortality, left and right gonad surface area, cortex thickness and percentage of seminiferous tubules of left gonad of embryos of the domestic fowl ( Gallus g. domesticus ). Effects of in ovo exposure to tamoxifen (TAM, 0.001, 0.01, 0.1, 1, 10 µg/g egg) and 17 α -ethinylestradiol (EE 2 , 20 ng/g egg) on mortality (A), left and right gonad surface area (B) and cortex thickness and percentage of seminiferous tubules of left gonad (C) of embryos of the domestic fowl ( Gallus g. domesticus ) on embryonic day 19. Statistical analysis by Fisher’s exact test (A), one-way ANOVA with Dunnett’s multiple comparisons test or Kruskal-Wallis test with Dunn’s multiple comparison test (B, C). Grey background distinguishes the co-exposure to tamoxifen and EE 2 . Lowercase indicates significant differences compared to the solvent control. Level of significance: a, p
    Figure Legend Snippet: Effects of in ovo exposure to tamoxifen and 17 α -ethinylestradiol on mortality, left and right gonad surface area, cortex thickness and percentage of seminiferous tubules of left gonad of embryos of the domestic fowl ( Gallus g. domesticus ). Effects of in ovo exposure to tamoxifen (TAM, 0.001, 0.01, 0.1, 1, 10 µg/g egg) and 17 α -ethinylestradiol (EE 2 , 20 ng/g egg) on mortality (A), left and right gonad surface area (B) and cortex thickness and percentage of seminiferous tubules of left gonad (C) of embryos of the domestic fowl ( Gallus g. domesticus ) on embryonic day 19. Statistical analysis by Fisher’s exact test (A), one-way ANOVA with Dunnett’s multiple comparisons test or Kruskal-Wallis test with Dunn’s multiple comparison test (B, C). Grey background distinguishes the co-exposure to tamoxifen and EE 2 . Lowercase indicates significant differences compared to the solvent control. Level of significance: a, p

    Techniques Used: In Ovo

    Right and left ovary of genetic females of untreated control group and tamoxifen-treated group on day 19 of embryonic development. Right and left ovary of genetic females of untreated control group (A, B, C) and tamoxifen-treated group (10 µg/g egg; D, E, F). (A, D) Unfixed right (“Or”) and left (“Ol”) ovary (outlined in black) on day 19 of embryonic development. Untreated control females (A) show a regressed right ovary and a well-differentiated left ovary. Tamoxifen-treated females (D) show an unaffected right ovary and a significantly decreased left ovary. (B, E) Histological thin sections (6 µm) of right (“Or”) and left (“Ol”) ovary. Untreated control females (B) show a left ovary of female typical size and structure. The left ovarian cortex (“C”) is well-differentiated and covers almost the whole ovary (except the region close to the mesonephron (“Me”)). Tamoxifen-treated females (E) show a significantly decreased left ovary with an altered distribution of the cortex region (“C”). The left ovary is no longer covered by a continuous cortex but exposes large regions of uncovered medulla, partly resembling a male testis. (C, F) Histological thin sections (6 µm) of the left ovary in close-up. Untreated control females (C) show a well-differentiated continuous ovarian cortex region (“C”) and a loosely arranged medulla (“M”) crossed by lacunar channels (“L”). Tamoxifen-treated females (F) show a discontinuous irregular scattered ovarian cortex region (“C”) with larger regions of uncovered medulla (“M”) crossed by lacunar channels (“L”).
    Figure Legend Snippet: Right and left ovary of genetic females of untreated control group and tamoxifen-treated group on day 19 of embryonic development. Right and left ovary of genetic females of untreated control group (A, B, C) and tamoxifen-treated group (10 µg/g egg; D, E, F). (A, D) Unfixed right (“Or”) and left (“Ol”) ovary (outlined in black) on day 19 of embryonic development. Untreated control females (A) show a regressed right ovary and a well-differentiated left ovary. Tamoxifen-treated females (D) show an unaffected right ovary and a significantly decreased left ovary. (B, E) Histological thin sections (6 µm) of right (“Or”) and left (“Ol”) ovary. Untreated control females (B) show a left ovary of female typical size and structure. The left ovarian cortex (“C”) is well-differentiated and covers almost the whole ovary (except the region close to the mesonephron (“Me”)). Tamoxifen-treated females (E) show a significantly decreased left ovary with an altered distribution of the cortex region (“C”). The left ovary is no longer covered by a continuous cortex but exposes large regions of uncovered medulla, partly resembling a male testis. (C, F) Histological thin sections (6 µm) of the left ovary in close-up. Untreated control females (C) show a well-differentiated continuous ovarian cortex region (“C”) and a loosely arranged medulla (“M”) crossed by lacunar channels (“L”). Tamoxifen-treated females (F) show a discontinuous irregular scattered ovarian cortex region (“C”) with larger regions of uncovered medulla (“M”) crossed by lacunar channels (“L”).

    Techniques Used:

    37) Product Images from "Wnt/β-catenin signaling regulates ependymal cell development and adult homeostasis"

    Article Title: Wnt/β-catenin signaling regulates ependymal cell development and adult homeostasis

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1803297115

    Wnt/β-catenin signaling is required for ependymal proliferation in the postnatal and adult spinal cord. ( A and D ) Experimental schedule used for β-catenin conditional knockout experiments. ( B ) Representative Ki67 (red) DAPI (blue) immunostaining images of spinal cords from Axin2 CreERT2/+ ; β-catenin fl/+ (control) and mutant Axin2 CreERT2/+ ; β-catenin fl/del (β-cat KO) littermates that received tamoxifen at P16. (Scale bar, 50 um.) ( C ) The percentages of Ki67 + ependymal cells out of total ependymal cells in control and KO animals. Control, n = 3; β-cat KO, n = 3. ( E ) Representative EdU (red) Vimentin (Yellow), and DAPI (blue) immunostaining images of spinal cords from Axin2 CreERT2/+ ; β-catenin fl/+ (control) and mutant Axin2 CreERT2/+ ; β-catenin fl/del (β-cat KO) littermates that received tamoxifen at P56–P60. (Scale bar, 50 um.) ( F ) The percentages of Ki67 + ependymal cells out of total ependymal cells in control and KO animals. Control, n = 4; β-cat KO, n = 4. ( G ) The percentages of EdU + ependymal cells out of total ependymal cells in control and KO animals. * P
    Figure Legend Snippet: Wnt/β-catenin signaling is required for ependymal proliferation in the postnatal and adult spinal cord. ( A and D ) Experimental schedule used for β-catenin conditional knockout experiments. ( B ) Representative Ki67 (red) DAPI (blue) immunostaining images of spinal cords from Axin2 CreERT2/+ ; β-catenin fl/+ (control) and mutant Axin2 CreERT2/+ ; β-catenin fl/del (β-cat KO) littermates that received tamoxifen at P16. (Scale bar, 50 um.) ( C ) The percentages of Ki67 + ependymal cells out of total ependymal cells in control and KO animals. Control, n = 3; β-cat KO, n = 3. ( E ) Representative EdU (red) Vimentin (Yellow), and DAPI (blue) immunostaining images of spinal cords from Axin2 CreERT2/+ ; β-catenin fl/+ (control) and mutant Axin2 CreERT2/+ ; β-catenin fl/del (β-cat KO) littermates that received tamoxifen at P56–P60. (Scale bar, 50 um.) ( F ) The percentages of Ki67 + ependymal cells out of total ependymal cells in control and KO animals. Control, n = 4; β-cat KO, n = 4. ( G ) The percentages of EdU + ependymal cells out of total ependymal cells in control and KO animals. * P

    Techniques Used: Knock-Out, Immunostaining, Mutagenesis

    Dorsal midline neural progenitor cells are Wnt responsive throughout spinal cord development. ( A ) Histologic X-gal stained sections of spinal cord from E10.5 Axin2-LacZ mice counterstained with Nuclear Fast Red. Arrow indicates Axin2-LacZ + cells. ( B ) Histologic X-gal stained sections of spinal cord from E17.5 Axin2-LacZ mice counterstained with Nuclear Fast Red. Axin2-LacZ + cells concentrate along the dorsal midline. Arrow indicates Axin2-LacZ + cells. ( C ) The central canal of the spinal cord from E17.5 TCF/Lef:H2B-GFP. GFP + cells concentrate along the dorsal midline. Dotted line outlines the central canal. Asterisk indicates the gap at the dorsalmost portion. ( D ) Cross-sectional image of Axin2 CreERT2/+ ; Rosa26 mTmG/+ spinal cord labeled at E8.5 and analyzed at E10.5. Axin2 CreERT2 marks dorsal midline neuroepithelial cells. Arrow indicates GFP + neuroepithelial cells. Arrowheads indicate GFP + neural crest cells. ( E and F ) Cross-sectional images of Axin2 CreERT2/+ ; Rosa26 mTmG/+ spinal cord labeled at E12.5 and analyzed at E14.5 ( E ), or labeled at E14.5 and analyzed at E16.5 ( F ). GFP + radial glial cells span the entire dorsal midline. ( G ) Dorsal midline radial glial cells retain radial processes that span the entire length from the central canal to the dorsal pial surface and are labeled with GFP upon tamoxifen administration at E17.5. ( H ) GFP + radial glial cells labeled at E17.5 and analyzed at P0 express Vimentin. (Scale bar, 50 µm.)
    Figure Legend Snippet: Dorsal midline neural progenitor cells are Wnt responsive throughout spinal cord development. ( A ) Histologic X-gal stained sections of spinal cord from E10.5 Axin2-LacZ mice counterstained with Nuclear Fast Red. Arrow indicates Axin2-LacZ + cells. ( B ) Histologic X-gal stained sections of spinal cord from E17.5 Axin2-LacZ mice counterstained with Nuclear Fast Red. Axin2-LacZ + cells concentrate along the dorsal midline. Arrow indicates Axin2-LacZ + cells. ( C ) The central canal of the spinal cord from E17.5 TCF/Lef:H2B-GFP. GFP + cells concentrate along the dorsal midline. Dotted line outlines the central canal. Asterisk indicates the gap at the dorsalmost portion. ( D ) Cross-sectional image of Axin2 CreERT2/+ ; Rosa26 mTmG/+ spinal cord labeled at E8.5 and analyzed at E10.5. Axin2 CreERT2 marks dorsal midline neuroepithelial cells. Arrow indicates GFP + neuroepithelial cells. Arrowheads indicate GFP + neural crest cells. ( E and F ) Cross-sectional images of Axin2 CreERT2/+ ; Rosa26 mTmG/+ spinal cord labeled at E12.5 and analyzed at E14.5 ( E ), or labeled at E14.5 and analyzed at E16.5 ( F ). GFP + radial glial cells span the entire dorsal midline. ( G ) Dorsal midline radial glial cells retain radial processes that span the entire length from the central canal to the dorsal pial surface and are labeled with GFP upon tamoxifen administration at E17.5. ( H ) GFP + radial glial cells labeled at E17.5 and analyzed at P0 express Vimentin. (Scale bar, 50 µm.)

    Techniques Used: Staining, Mouse Assay, Labeling

    38) Product Images from "HSPB7 prevents cardiac conduction system defect through maintaining intercalated disc integrity"

    Article Title: HSPB7 prevents cardiac conduction system defect through maintaining intercalated disc integrity

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1006984

    Characterization of HSPB7 CKO mouse. (A) Immunoblot analysis for HSPB7 protein expression levels in control (Flox/Flox) and CKO animals 4 days (d4) and 7 days (d7) after tamoxifen administration. The GAPDH signal shows the loading of samples between lanes. (B) Quantitative analysis for immunoblot analysis of HSPB7 expression protein levels in cardiac tissue from control and CKO mice. Seven days after the first tamoxifen administration, HSPB7 protein expression dropped to less than 10% in CKO animals, as determined by immunoblot blot (HSPB7 compared with GAPDH). Data are presented as means ± SD. (C) Double staining of the left ventricle section with HSPB7 and Hoechst 33342 in the control and CKO hearts at d7 after tamoxifen administration. HSPB7 was no longer present at the intercalated disc and sarcomere. Scale bar: 20 μm (D) Significant increase in heart weight in HSPB7 CKO mice (n = 8; * p = 0.00027). (E) Kaplan–Meier survival curve of HSPB7 CKO and control mice. (F) Representative whole mounts (left), Masson’s trichrome (middle left and right), and hematoxylin-eosin–stained (right) transverse sections of control and CKO mouse hearts. Histological analysis showed inflammatory infiltration in the myocardium, identified as mostly lymphocytes and plasma cells, in HSPB7 CKO hearts at d7 after tamoxifen administration. In addition, Masson’s trichrome staining showed no significant collagen deposition in HSPB7 CKO hearts, as compared to control mice.
    Figure Legend Snippet: Characterization of HSPB7 CKO mouse. (A) Immunoblot analysis for HSPB7 protein expression levels in control (Flox/Flox) and CKO animals 4 days (d4) and 7 days (d7) after tamoxifen administration. The GAPDH signal shows the loading of samples between lanes. (B) Quantitative analysis for immunoblot analysis of HSPB7 expression protein levels in cardiac tissue from control and CKO mice. Seven days after the first tamoxifen administration, HSPB7 protein expression dropped to less than 10% in CKO animals, as determined by immunoblot blot (HSPB7 compared with GAPDH). Data are presented as means ± SD. (C) Double staining of the left ventricle section with HSPB7 and Hoechst 33342 in the control and CKO hearts at d7 after tamoxifen administration. HSPB7 was no longer present at the intercalated disc and sarcomere. Scale bar: 20 μm (D) Significant increase in heart weight in HSPB7 CKO mice (n = 8; * p = 0.00027). (E) Kaplan–Meier survival curve of HSPB7 CKO and control mice. (F) Representative whole mounts (left), Masson’s trichrome (middle left and right), and hematoxylin-eosin–stained (right) transverse sections of control and CKO mouse hearts. Histological analysis showed inflammatory infiltration in the myocardium, identified as mostly lymphocytes and plasma cells, in HSPB7 CKO hearts at d7 after tamoxifen administration. In addition, Masson’s trichrome staining showed no significant collagen deposition in HSPB7 CKO hearts, as compared to control mice.

    Techniques Used: Expressing, Mouse Assay, Double Staining, Staining

    Echocardiographic measurements of HSPB7 CKO and control mice. (A) Representative two-dimensional and M-mode echocardiographic images of the HSPB7 CKO and control hearts. (B) Echocardiographic analysis in the HSPB7 CKO and control mice before and at 4 days (d4) and 7 days (d7) after tamoxifen administration. Left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-systolic internal diameter (LVIDs), and left ventricular end-diastolic internal diameter (LVIDd). Data are means ± SD; n = 5 per group. *, P
    Figure Legend Snippet: Echocardiographic measurements of HSPB7 CKO and control mice. (A) Representative two-dimensional and M-mode echocardiographic images of the HSPB7 CKO and control hearts. (B) Echocardiographic analysis in the HSPB7 CKO and control mice before and at 4 days (d4) and 7 days (d7) after tamoxifen administration. Left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-systolic internal diameter (LVIDs), and left ventricular end-diastolic internal diameter (LVIDd). Data are means ± SD; n = 5 per group. *, P

    Techniques Used: Mouse Assay

    Electrical conduction is impaired in HSPB7 CKO hearts. (A and B) Annotated ECG curve of the HSPB7 CKO and control animals before (A) and 7 days after (B) the first tamoxifen injection. (C and D) Quantification of the ECG changes in HSPB7 CKO and control animals before (C) and 7 days after (D) the first tamoxifen administration. N = 5 per group. Data are presented as means ± SD. *, P
    Figure Legend Snippet: Electrical conduction is impaired in HSPB7 CKO hearts. (A and B) Annotated ECG curve of the HSPB7 CKO and control animals before (A) and 7 days after (B) the first tamoxifen injection. (C and D) Quantification of the ECG changes in HSPB7 CKO and control animals before (C) and 7 days after (D) the first tamoxifen administration. N = 5 per group. Data are presented as means ± SD. *, P

    Techniques Used: Injection

    Expression and localization of ID complex proteins in HSPB7 CKO hearts. (A) Confocal micrographs of longitudinal sections of the cardiac muscle of control and CKO mice at d7 after tamoxifen administration. Specific antibodies were used to identify the distributions of intercalated disc components: N-cadherin, desmoplakin, vinculin, and connexin 43. In HSPB7 CKO hearts, the staining of desmoplakin and N-cadherin were distributed throughout the cytoplasm, but little was observed in control hearts. Connexin 43 was absent from the intercalated discs in HSPB7 CKO hearts. The insets show representative areas at a higher magnification. The nucleus was visualized through Hoechst 33342 staining. n = 4 per group. Scale bar: 20 μm. (B) Immunoblotting of intercalated discs and sarcomeric-associated proteins in HSPB7 CKO and control hearts. GAPDH signal shows the loading of the samples between the lanes. n = 4 per group. (C) Quantitative analysis of immunoblot analysis of protein levels in cardiac tissue from control and CKO mice. Seven days after the first tamoxifen injections, connexin 43 protein expression dropped to
    Figure Legend Snippet: Expression and localization of ID complex proteins in HSPB7 CKO hearts. (A) Confocal micrographs of longitudinal sections of the cardiac muscle of control and CKO mice at d7 after tamoxifen administration. Specific antibodies were used to identify the distributions of intercalated disc components: N-cadherin, desmoplakin, vinculin, and connexin 43. In HSPB7 CKO hearts, the staining of desmoplakin and N-cadherin were distributed throughout the cytoplasm, but little was observed in control hearts. Connexin 43 was absent from the intercalated discs in HSPB7 CKO hearts. The insets show representative areas at a higher magnification. The nucleus was visualized through Hoechst 33342 staining. n = 4 per group. Scale bar: 20 μm. (B) Immunoblotting of intercalated discs and sarcomeric-associated proteins in HSPB7 CKO and control hearts. GAPDH signal shows the loading of the samples between the lanes. n = 4 per group. (C) Quantitative analysis of immunoblot analysis of protein levels in cardiac tissue from control and CKO mice. Seven days after the first tamoxifen injections, connexin 43 protein expression dropped to

    Techniques Used: Expressing, Mouse Assay, Staining

    Loss of HSPB7 results in FLNC protein upregulation and aggregation. Confocal micrographs of longitudinal sections (A) and cross-sections (B) of the heart. FLNC aggregation (arrowheads) was observed only in the mutant cardiomyocytes. Extracellular matrix accumulation was labeled using wheat germ agglutinin (WGA). The nucleus was visualized through Hoechst 33342 staining. Scale bar: 50 μm. (C) Immunoblot analysis and quantitation (D) of the expression levels of FLNC in the cardiac muscle. The muscle homogenate supernatant (S) and pellet (P) fractions were analyzed from control and CKO mice at d7 after tamoxifen administration. GAPDH and Coomassie Blue staining were used to verify the loading amount in the supernatant and pellet. n = 3 per group. Data are presented as means ± SD. *, P
    Figure Legend Snippet: Loss of HSPB7 results in FLNC protein upregulation and aggregation. Confocal micrographs of longitudinal sections (A) and cross-sections (B) of the heart. FLNC aggregation (arrowheads) was observed only in the mutant cardiomyocytes. Extracellular matrix accumulation was labeled using wheat germ agglutinin (WGA). The nucleus was visualized through Hoechst 33342 staining. Scale bar: 50 μm. (C) Immunoblot analysis and quantitation (D) of the expression levels of FLNC in the cardiac muscle. The muscle homogenate supernatant (S) and pellet (P) fractions were analyzed from control and CKO mice at d7 after tamoxifen administration. GAPDH and Coomassie Blue staining were used to verify the loading amount in the supernatant and pellet. n = 3 per group. Data are presented as means ± SD. *, P

    Techniques Used: Mutagenesis, Labeling, Whole Genome Amplification, Staining, Quantitation Assay, Expressing, Mouse Assay

    Ultrastructural study of control and HSPB7 CKO hearts. Transmission electron micrographs (TEMs) of ventricular myocardium from HSPB7 CKO and control mice at d7 after tamoxifen administration. Right panels are higher-magnification views of the boxed areas in the left panels. (A) Normal intercalated disc structures were visible in the control hearts. The inset provides a simplistic representation of the morphology of the intercalated discs. Higher-magnification images showed abnormal adherens junctions (black arrowheads) and desmosomes (white arrowheads) with widened gaps of the intercalated discs in HSPB7 mutant hearts. Abnormal Z-line (black arrow), filament disruption (asterisk), and detachment of myofibrils at the intercalated disc (white arrow) were also observed in the CKO mice. (B) The ultrastructure of the sarcomeres at the center part of the cardiomyocyte was slightly distorted. Higher-magnification images showed (right panel) loose actin filaments (black arrowhead) and wider, less dense Z-lines (white arrowhead) compared with the controls. n = 3 per group. Scale bar: 250 nm.
    Figure Legend Snippet: Ultrastructural study of control and HSPB7 CKO hearts. Transmission electron micrographs (TEMs) of ventricular myocardium from HSPB7 CKO and control mice at d7 after tamoxifen administration. Right panels are higher-magnification views of the boxed areas in the left panels. (A) Normal intercalated disc structures were visible in the control hearts. The inset provides a simplistic representation of the morphology of the intercalated discs. Higher-magnification images showed abnormal adherens junctions (black arrowheads) and desmosomes (white arrowheads) with widened gaps of the intercalated discs in HSPB7 mutant hearts. Abnormal Z-line (black arrow), filament disruption (asterisk), and detachment of myofibrils at the intercalated disc (white arrow) were also observed in the CKO mice. (B) The ultrastructure of the sarcomeres at the center part of the cardiomyocyte was slightly distorted. Higher-magnification images showed (right panel) loose actin filaments (black arrowhead) and wider, less dense Z-lines (white arrowhead) compared with the controls. n = 3 per group. Scale bar: 250 nm.

    Techniques Used: Transmission Assay, Mouse Assay, Mutagenesis

    39) Product Images from "Coregulator-dependent facilitation of chromatin occupancy by GATA-1"

    Article Title: Coregulator-dependent facilitation of chromatin occupancy by GATA-1

    Journal:

    doi: 10.1073/pnas.0307612100

    FOG-1 occupies sites in which the FOG-1-dependent GATA switches occur. Quantitative ChIP analysis was conducted with anti-FOG-1 antibody in FOG-1 –/– , G1E, and tamoxifen-treated G1E–ER–GATA-1 cells. ( A ) The graph depicts
    Figure Legend Snippet: FOG-1 occupies sites in which the FOG-1-dependent GATA switches occur. Quantitative ChIP analysis was conducted with anti-FOG-1 antibody in FOG-1 –/– , G1E, and tamoxifen-treated G1E–ER–GATA-1 cells. ( A ) The graph depicts

    Techniques Used: Chromatin Immunoprecipitation

    High-level overexpression of ER–GATA-1 does not efficiently repress GATA - 2 transcription in FOG-1 –/– cells. ( A ) Western blot analysis of GATA-1 and ER–GATA-1 expression in whole cell lysates from untreated and tamoxifen-treated
    Figure Legend Snippet: High-level overexpression of ER–GATA-1 does not efficiently repress GATA - 2 transcription in FOG-1 –/– cells. ( A ) Western blot analysis of GATA-1 and ER–GATA-1 expression in whole cell lysates from untreated and tamoxifen-treated

    Techniques Used: Over Expression, Western Blot, Expressing

    40) Product Images from "Adult AMPA GLUA1 Receptor Subunit Loss in 5-HT Neurons Results in a Specific Anxiety-Phenotype with Evidence for Dysregulation of 5-HT Neuronal Activity"

    Article Title: Adult AMPA GLUA1 Receptor Subunit Loss in 5-HT Neurons Results in a Specific Anxiety-Phenotype with Evidence for Dysregulation of 5-HT Neuronal Activity

    Journal: Neuropsychopharmacology

    doi: 10.1038/npp.2014.332

    Immunohistochemical verification of 5-HT neuron-specific GLUA1 loss. Brains of Gria1 5-HT−/− and Gria1 fl/fl mice were removed 4 weeks after tamoxifen administration. Double-fluorescent IHC with TPH2- (green) and GLUA1-antibodies (red) was performed, as shown in color-separated images and in the corresponding overlay (a3–f3) in the dorsal (a1–d3) and median (e1–f3) raphe nucleus. White boxes indicate the tissue areas enlarged in the insets. (a1–b3, e1–e3) In Gria1 fl/fl mice, most immunopositive cells for TPH2 in both the median and dorsal raphe nucleus co-expressed GLUA1. Labeling for TPH2 was distributed in cell bodies and dendrites, while labeling for GLUA1 was mostly restricted to the neuropil but also found in the cytoplasm of neurons (f.i. white arrows in b1–b3 and e1–e3). (c1–d3, f1–f3) In Gria1 5-HT−/− mice, most TPH2-immunopositive cells in both the median and dorsal raphe nucleus were immunonegative for GLUA1. Labeling for TPH2 was distributed in cell bodies and dendrites, while labeling for GLUA1 was absent from cell bodies (f.i. white asterisks in d2) and only detectable in the neuropil. Scale bar: a, c, e, f: 100 μm; b, d: 50 μm.
    Figure Legend Snippet: Immunohistochemical verification of 5-HT neuron-specific GLUA1 loss. Brains of Gria1 5-HT−/− and Gria1 fl/fl mice were removed 4 weeks after tamoxifen administration. Double-fluorescent IHC with TPH2- (green) and GLUA1-antibodies (red) was performed, as shown in color-separated images and in the corresponding overlay (a3–f3) in the dorsal (a1–d3) and median (e1–f3) raphe nucleus. White boxes indicate the tissue areas enlarged in the insets. (a1–b3, e1–e3) In Gria1 fl/fl mice, most immunopositive cells for TPH2 in both the median and dorsal raphe nucleus co-expressed GLUA1. Labeling for TPH2 was distributed in cell bodies and dendrites, while labeling for GLUA1 was mostly restricted to the neuropil but also found in the cytoplasm of neurons (f.i. white arrows in b1–b3 and e1–e3). (c1–d3, f1–f3) In Gria1 5-HT−/− mice, most TPH2-immunopositive cells in both the median and dorsal raphe nucleus were immunonegative for GLUA1. Labeling for TPH2 was distributed in cell bodies and dendrites, while labeling for GLUA1 was absent from cell bodies (f.i. white asterisks in d2) and only detectable in the neuropil. Scale bar: a, c, e, f: 100 μm; b, d: 50 μm.

    Techniques Used: Immunohistochemistry, Mouse Assay, Labeling

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    Article Snippet: .. For in vivo genetic lineage-tracing experiments, tamoxifen (Sigma, T5648) was resuspended in ethanol and diluted in corn oil at a concentration of 10 mg/mL and administered via intra-peritoneal injections daily for 4 consecutive days using the following regimen: 3mg, 2mg, 2mg, 2mg. .. Isolation of mouse prostate cellsAll dissections were performed under a stereo microscope in sterile PBS.

    Mouse Assay:

    Article Title: Wnt-mediated interactions of tumor-initiating cells with a macrophage niche drive skin tumor formation
    Article Snippet: .. 5 weeks-old Cd68CreERT2;Wlslox/lox mice were treated with tamoxifen (Sigma-Aldrich) (10 mg/mL dissolved in sunflower seed oil, 2 mg per day) via i.p. during 4 consecutive days before the DMBA-TPA treatment, and through the duration of the experiments, tamoxifen was administered orally in the pellet food. ..

    Article Title: Resident c-kit+ cells in the heart are not cardiac stem cells
    Article Snippet: .. Tamoxifen (Sigma, Cat. T5648) was intraperitoneally injected into mice (0.12 mg g−1 body weight). ..

    Article Title: Evidence That Dendritic Mitochondria Negatively Regulate Dendritic Branching in Pyramidal Neurons in the Neocortex
    Article Snippet: .. Tamoxifen (Sigma) was dissolved in corn oil (Sigma) at 10 mg/ml and administered by oral gavage to pregnant mice (5 mg per animal) at E18.5–18.75. ..

    Concentration Assay:

    Article Title: Sonic hedgehog signaling in astrocytes mediates cell type-specific synaptic organization
    Article Snippet: .. Briefly, tamoxifen (Sigma, T5648) was dissolved in corn oil to a final concentration of 20 mg/ml. .. Adult Gli1 CreER /+ ;Ai14 mice received 250mg/kg tamoxifen by oral gavage for three consecutive days, and tissues were analyzed two – three weeks later.

    Article Title: Off-Target Effects in Transgenic Mice: Characterization of Dopamine Transporter (DAT)-Cre Transgenic Mouse Lines Exposes Multiple Non-Dopaminergic Neuronal Clusters Available for Selective Targeting within Limbic Neurocircuitry
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    Expressing:

    Article Title: An Immunocompetent Mouse Model of HPV16(+) Head and Neck Squamous Cell Carcinoma
    Article Snippet: .. Tamoxifen and 4NQO administration To conditionally induce GpNLuc or E7iresE6 expression, iLumiFluor or iKH and iKHP animals were intra -lingually injected using a 27-gauge needle with 0.5mg of tamoxifen (Sigma-Aldrich, cat. #: T5648) dissolved in 30µl corn oil (MP Biomedical, cat. #: 901414) either two and/or three times over a five-day period, respectively. .. 4NQO (4-Nitroquinoline 1-oxide; Sigma, cat. #: N8141) was dissolved in propylene glycol (Sigma, cat. #: 398039) at a concentration of 5 mg/mL and then further diluted to a final concentration of 10 or 20 µg/ml in sterile ddH2O.

    Sonication:

    Article Title: Stability and function of regulatory T cells expressing the transcription factor T-bet
    Article Snippet: .. For tamoxifen administration, 40 mg tamoxifen dissolved in 100 μL olive oil (Sigma-Aldrich) were sonicated 4 × 30 seconds in a Bioruptor Twin (Diagenode). .. Mice were orally gavaged with 200μl tamoxifen emulsion per treatment.

    Injection:

    Article Title: Resident c-kit+ cells in the heart are not cardiac stem cells
    Article Snippet: .. Tamoxifen (Sigma, Cat. T5648) was intraperitoneally injected into mice (0.12 mg g−1 body weight). ..

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  • 95
    Millipore tamoxifen citrate
    Enhancement of <t>tamoxifen</t> sensitivity by NF-κB inhibition. (a) To determine the mechanisms of NF-κB-ER crosstalk, the expression levels of ERα were detected. The original MCF-7 cells were cultured in serum free and phenol red free AIM-V, and hormon-independent MCF-7 subline was obtained. Whole cell lysates were obtained from hormon-independent MCF-7 subline that was incubated in the indicated concentrations of IMD-0354 for 7 days, and then the ERα proteins were detected by Western blot analysis. These cropped blots represented data from 3 individual blots are used in the main figures and full-length blots are included in the supplementary information ( Supplementary Fig. S8 ). Each value in the graph represents the mean ± SE of 3 different experiments; *, P
    Tamoxifen Citrate, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tamoxifen citrate/product/Millipore
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    tamoxifen citrate - by Bioz Stars, 2020-09
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    99
    Millipore rhobtb1
    Protective effect of <t>RhoBTB1</t> on Ang-II–induced hypertension. ( A and B ) Relative mRNA expression of RhoBTB1 was determined by qPCR in aorta from control ( A ) or S-RhoBTB1 ( B ) mice after injection of vehicle (corn oil) or Tx and subsequent treatment with Ang-II (490 ng/min/kg) or saline for 14 days. ( C and D ) Systolic BP (SBP) was measured for 14 days after initiation of Ang-II infusion (arrow) in control ( C ) and S-RhoBTB1 ( D ) mice. Sample numbers are indicated in E . ( E ) Cumulative SBP during the last 4 days of measurement from mice in C and D . ( F – K ) Cumulative concentration-response curves for ACh ( F – I ) and SNP ( J and K ) in aorta ( n = 5–8) and basilar artery ( n = 4) ( H – I ). ( L and M ) Cumulative concentration-response curves for 5-HT. ( N ) Mutations in either Cullin-3 or PPARγ cause hypertension. Cullin-3 regulates both the Rho/ROCK pathway through BACURD, whereas Cullin-3 regulates PDE5 through RhoBTB1. RhoBTB1 regulates the activity of PDE5 by ensuring that excess PDE5 is targeted for Cullin-3 dependent ubiquitination and proteasomal degradation. Mutations in PPARγ or treatment with Ang-II causes RhoBTB1-deficiency. All data represent the mean ± SEM. * P
    Rhobtb1, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 387 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rhobtb1/product/Millipore
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    rhobtb1 - by Bioz Stars, 2020-09
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    Enhancement of tamoxifen sensitivity by NF-κB inhibition. (a) To determine the mechanisms of NF-κB-ER crosstalk, the expression levels of ERα were detected. The original MCF-7 cells were cultured in serum free and phenol red free AIM-V, and hormon-independent MCF-7 subline was obtained. Whole cell lysates were obtained from hormon-independent MCF-7 subline that was incubated in the indicated concentrations of IMD-0354 for 7 days, and then the ERα proteins were detected by Western blot analysis. These cropped blots represented data from 3 individual blots are used in the main figures and full-length blots are included in the supplementary information ( Supplementary Fig. S8 ). Each value in the graph represents the mean ± SE of 3 different experiments; *, P

    Journal: Scientific Reports

    Article Title: Nuclear factor-?B plays a critical role in both intrinsic and acquired resistance against endocrine therapy in human breast cancer cells

    doi: 10.1038/srep04057

    Figure Lengend Snippet: Enhancement of tamoxifen sensitivity by NF-κB inhibition. (a) To determine the mechanisms of NF-κB-ER crosstalk, the expression levels of ERα were detected. The original MCF-7 cells were cultured in serum free and phenol red free AIM-V, and hormon-independent MCF-7 subline was obtained. Whole cell lysates were obtained from hormon-independent MCF-7 subline that was incubated in the indicated concentrations of IMD-0354 for 7 days, and then the ERα proteins were detected by Western blot analysis. These cropped blots represented data from 3 individual blots are used in the main figures and full-length blots are included in the supplementary information ( Supplementary Fig. S8 ). Each value in the graph represents the mean ± SE of 3 different experiments; *, P

    Article Snippet: Because those sublines exhibited a non-adherent phenotype and formed spheres , for the evaluation of cell growth activities, original cells (1 × 105 cells/mL) were incubated with IMD-0354 or tamoxifen citrate (Millipore) in serum-free media for 7 days, and then the number and size of obtained spheres were measured.

    Techniques: Inhibition, Expressing, Cell Culture, Incubation, Radial Immuno Diffusion, Western Blot

    Tamoxifen does not affect normal locomotor activity in rats. Locomotor activity of male Sprague-Dawley rats was monitored for 2 hours before and 2 hours after intraperitoneal injection. On days 1 and 2, animals received either 5 mg/kg tamoxifen citrate or an equivalent volume of vehicle. On day 3, all animals received saline. Data are represented as the mean ± S.E.M. ( n = 8). Hab, XXX; Sal, saline; TMX, tamoxifen; Veh, vehicle.

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    Article Title: Tamoxifen Directly Interacts with the Dopamine Transporter

    doi: 10.1124/jpet.118.248179

    Figure Lengend Snippet: Tamoxifen does not affect normal locomotor activity in rats. Locomotor activity of male Sprague-Dawley rats was monitored for 2 hours before and 2 hours after intraperitoneal injection. On days 1 and 2, animals received either 5 mg/kg tamoxifen citrate or an equivalent volume of vehicle. On day 3, all animals received saline. Data are represented as the mean ± S.E.M. ( n = 8). Hab, XXX; Sal, saline; TMX, tamoxifen; Veh, vehicle.

    Article Snippet: Tamoxifen citrate was dissolved in 100% dimethylsulfoxide (DMSO) at a concentration of 50 mM and diluted further for in vitro studies, or at 2 mg/ml in 3.5% DMSO and 10% Tween 80 in saline for in vivo studies.

    Techniques: Activity Assay, Injection

    Tamoxifen stabilizes the outward-facing conformation of the DAT in a cocaine-like manner. Rat striatal synaptosomes were incubated for 1 hour with 100 μ M cocaine, 10 μ M tamoxifen, or vehicle prior to biotinylation of surface cysteines with maleimide-PEG 2 -biotin. DAT content in biotinylated fractions was quantified by Western blotting. (A) Biotinylated transporter/total transporter in lysate. * P

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    Article Title: Tamoxifen Directly Interacts with the Dopamine Transporter

    doi: 10.1124/jpet.118.248179

    Figure Lengend Snippet: Tamoxifen stabilizes the outward-facing conformation of the DAT in a cocaine-like manner. Rat striatal synaptosomes were incubated for 1 hour with 100 μ M cocaine, 10 μ M tamoxifen, or vehicle prior to biotinylation of surface cysteines with maleimide-PEG 2 -biotin. DAT content in biotinylated fractions was quantified by Western blotting. (A) Biotinylated transporter/total transporter in lysate. * P

    Article Snippet: Tamoxifen citrate was dissolved in 100% dimethylsulfoxide (DMSO) at a concentration of 50 mM and diluted further for in vitro studies, or at 2 mg/ml in 3.5% DMSO and 10% Tween 80 in saline for in vivo studies.

    Techniques: Incubation, Western Blot

    Tamoxifen attenuates amphetamine-stimulated dopamine efflux. Striatal synaptosomes were perfused for 1 hour at 37°C with vehicle or various concentrations of tamoxifen as described in the Materials and Methods . Amphetamine (10 μ M) was included in the perfusate during fractions 7 and 8. Data were calculated as the area under the curve after treatment with amphetamine. Data are represented as the mean ± S.E.M. ( n = 3–5). * P

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    Article Title: Tamoxifen Directly Interacts with the Dopamine Transporter

    doi: 10.1124/jpet.118.248179

    Figure Lengend Snippet: Tamoxifen attenuates amphetamine-stimulated dopamine efflux. Striatal synaptosomes were perfused for 1 hour at 37°C with vehicle or various concentrations of tamoxifen as described in the Materials and Methods . Amphetamine (10 μ M) was included in the perfusate during fractions 7 and 8. Data were calculated as the area under the curve after treatment with amphetamine. Data are represented as the mean ± S.E.M. ( n = 3–5). * P

    Article Snippet: Tamoxifen citrate was dissolved in 100% dimethylsulfoxide (DMSO) at a concentration of 50 mM and diluted further for in vitro studies, or at 2 mg/ml in 3.5% DMSO and 10% Tween 80 in saline for in vivo studies.

    Techniques:

    Tamoxifen readily docks in the S2 pocket of hDAT modeled after LeuT. The in silico hDAT model based on LeuT was constructed as described in the Materials and Methods . (A) Final energy-minimized pose of DAT/tamoxifen complex after flexible docking of tamoxifen at the DAT secondary (S2) substrate binding site. Selected binding pocket residues are labeled and rendered as sticks; bound tamoxifen (also shown in sticks) is highlighted in yellow. Cotransported sodium and chloride atoms are labeled and rendered as orange and green spheres, respectively. (B) Two-dimensional interaction diagram of tamoxifen bound at the S2 site of DAT. The interaction map depicts respective DAT residues located within 4.5 Å of the bound tamoxifen molecule (hydrophobic residues are colored green and polar residues are purple). The most significant non–van der Waals DAT/ligand interactions are indicated with dotted lines and a symbol depicting the chemistry of the interaction formed: side-chain hydrogen bond between D476 and amine nitrogen of tamoxifen (green), and an aromatic H/π-bond interaction between one of the tamoxifen rings and W84 (green, hexagon H).

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    Article Title: Tamoxifen Directly Interacts with the Dopamine Transporter

    doi: 10.1124/jpet.118.248179

    Figure Lengend Snippet: Tamoxifen readily docks in the S2 pocket of hDAT modeled after LeuT. The in silico hDAT model based on LeuT was constructed as described in the Materials and Methods . (A) Final energy-minimized pose of DAT/tamoxifen complex after flexible docking of tamoxifen at the DAT secondary (S2) substrate binding site. Selected binding pocket residues are labeled and rendered as sticks; bound tamoxifen (also shown in sticks) is highlighted in yellow. Cotransported sodium and chloride atoms are labeled and rendered as orange and green spheres, respectively. (B) Two-dimensional interaction diagram of tamoxifen bound at the S2 site of DAT. The interaction map depicts respective DAT residues located within 4.5 Å of the bound tamoxifen molecule (hydrophobic residues are colored green and polar residues are purple). The most significant non–van der Waals DAT/ligand interactions are indicated with dotted lines and a symbol depicting the chemistry of the interaction formed: side-chain hydrogen bond between D476 and amine nitrogen of tamoxifen (green), and an aromatic H/π-bond interaction between one of the tamoxifen rings and W84 (green, hexagon H).

    Article Snippet: Tamoxifen citrate was dissolved in 100% dimethylsulfoxide (DMSO) at a concentration of 50 mM and diluted further for in vitro studies, or at 2 mg/ml in 3.5% DMSO and 10% Tween 80 in saline for in vivo studies.

    Techniques: In Silico, Construct, Binding Assay, Labeling

    Tamoxifen impairs dopamine uptake. (A and B) Synaptosomes from the striatum of male Sprague-Dawley rats were incubated for 1 hour at 37°C with vehicle or indicated concentrations of tamoxifen, then treated with 310 nM (A) or indicated concentrations of [ 3 H]DA and incubated for an additional 3 minutes (A) or 30 seconds (B). (A) Data are represented as the mean ± S.E.M. ( n = 5). * P

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    Article Title: Tamoxifen Directly Interacts with the Dopamine Transporter

    doi: 10.1124/jpet.118.248179

    Figure Lengend Snippet: Tamoxifen impairs dopamine uptake. (A and B) Synaptosomes from the striatum of male Sprague-Dawley rats were incubated for 1 hour at 37°C with vehicle or indicated concentrations of tamoxifen, then treated with 310 nM (A) or indicated concentrations of [ 3 H]DA and incubated for an additional 3 minutes (A) or 30 seconds (B). (A) Data are represented as the mean ± S.E.M. ( n = 5). * P

    Article Snippet: Tamoxifen citrate was dissolved in 100% dimethylsulfoxide (DMSO) at a concentration of 50 mM and diluted further for in vitro studies, or at 2 mg/ml in 3.5% DMSO and 10% Tween 80 in saline for in vivo studies.

    Techniques: Incubation

    Tamoxifen does not affect surface expression of the DAT. Rat striatal synaptosomes were incubated for 1 hour with 10 μ M tamoxifen or vehicle prior to biotinylation of surface proteins with sulfo-NHS-biotin. After avidin-biotin pulldown, DAT content in biotinylated fractions and lysates was quantified by Western blotting. (A) Biotinylated transporter/total transporter in lysate. (B) Representative Western blots showing the biotinylated fraction blotted for DAT protein and Na + /K + -ATPase, and the corresponding total lysate. Calculations of the ratio (± S.E.M.) of the optical densities of biotinylated transporter–Na + /K + -ATPase were 1.09 ± 0.2 for vehicle and 0.96 ± 0.1 for tamoxifen (data not shown) ( n = 3). TMX, tamoxifen; Veh, vehicle.

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    Article Title: Tamoxifen Directly Interacts with the Dopamine Transporter

    doi: 10.1124/jpet.118.248179

    Figure Lengend Snippet: Tamoxifen does not affect surface expression of the DAT. Rat striatal synaptosomes were incubated for 1 hour with 10 μ M tamoxifen or vehicle prior to biotinylation of surface proteins with sulfo-NHS-biotin. After avidin-biotin pulldown, DAT content in biotinylated fractions and lysates was quantified by Western blotting. (A) Biotinylated transporter/total transporter in lysate. (B) Representative Western blots showing the biotinylated fraction blotted for DAT protein and Na + /K + -ATPase, and the corresponding total lysate. Calculations of the ratio (± S.E.M.) of the optical densities of biotinylated transporter–Na + /K + -ATPase were 1.09 ± 0.2 for vehicle and 0.96 ± 0.1 for tamoxifen (data not shown) ( n = 3). TMX, tamoxifen; Veh, vehicle.

    Article Snippet: Tamoxifen citrate was dissolved in 100% dimethylsulfoxide (DMSO) at a concentration of 50 mM and diluted further for in vitro studies, or at 2 mg/ml in 3.5% DMSO and 10% Tween 80 in saline for in vivo studies.

    Techniques: Expressing, Incubation, Avidin-Biotin Assay, Western Blot

    Tamoxifen inhibits [ 3 H]WIN 35,428 binding to the DAT in rat striatal membranes. Rat striatal membranes were incubated with [ 3 H]WIN 35,428 with or without tamoxifen or vehicle for 3 hours at 4°C. Nonspecific binding was determined with 30 μ M nomifensine. (A) Membranes were incubated with 4 nM [ 3 H]WIN 35,428 without and with various concentrations of tamoxifen ( n = 8). ** P

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    Article Title: Tamoxifen Directly Interacts with the Dopamine Transporter

    doi: 10.1124/jpet.118.248179

    Figure Lengend Snippet: Tamoxifen inhibits [ 3 H]WIN 35,428 binding to the DAT in rat striatal membranes. Rat striatal membranes were incubated with [ 3 H]WIN 35,428 with or without tamoxifen or vehicle for 3 hours at 4°C. Nonspecific binding was determined with 30 μ M nomifensine. (A) Membranes were incubated with 4 nM [ 3 H]WIN 35,428 without and with various concentrations of tamoxifen ( n = 8). ** P

    Article Snippet: Tamoxifen citrate was dissolved in 100% dimethylsulfoxide (DMSO) at a concentration of 50 mM and diluted further for in vitro studies, or at 2 mg/ml in 3.5% DMSO and 10% Tween 80 in saline for in vivo studies.

    Techniques: Binding Assay, Incubation

    Protective effect of RhoBTB1 on Ang-II–induced hypertension. ( A and B ) Relative mRNA expression of RhoBTB1 was determined by qPCR in aorta from control ( A ) or S-RhoBTB1 ( B ) mice after injection of vehicle (corn oil) or Tx and subsequent treatment with Ang-II (490 ng/min/kg) or saline for 14 days. ( C and D ) Systolic BP (SBP) was measured for 14 days after initiation of Ang-II infusion (arrow) in control ( C ) and S-RhoBTB1 ( D ) mice. Sample numbers are indicated in E . ( E ) Cumulative SBP during the last 4 days of measurement from mice in C and D . ( F – K ) Cumulative concentration-response curves for ACh ( F – I ) and SNP ( J and K ) in aorta ( n = 5–8) and basilar artery ( n = 4) ( H – I ). ( L and M ) Cumulative concentration-response curves for 5-HT. ( N ) Mutations in either Cullin-3 or PPARγ cause hypertension. Cullin-3 regulates both the Rho/ROCK pathway through BACURD, whereas Cullin-3 regulates PDE5 through RhoBTB1. RhoBTB1 regulates the activity of PDE5 by ensuring that excess PDE5 is targeted for Cullin-3 dependent ubiquitination and proteasomal degradation. Mutations in PPARγ or treatment with Ang-II causes RhoBTB1-deficiency. All data represent the mean ± SEM. * P

    Journal: The Journal of Clinical Investigation

    Article Title: RhoBTB1 protects against hypertension and arterial stiffness by restraining phosphodiesterase 5 activity

    doi: 10.1172/JCI123462

    Figure Lengend Snippet: Protective effect of RhoBTB1 on Ang-II–induced hypertension. ( A and B ) Relative mRNA expression of RhoBTB1 was determined by qPCR in aorta from control ( A ) or S-RhoBTB1 ( B ) mice after injection of vehicle (corn oil) or Tx and subsequent treatment with Ang-II (490 ng/min/kg) or saline for 14 days. ( C and D ) Systolic BP (SBP) was measured for 14 days after initiation of Ang-II infusion (arrow) in control ( C ) and S-RhoBTB1 ( D ) mice. Sample numbers are indicated in E . ( E ) Cumulative SBP during the last 4 days of measurement from mice in C and D . ( F – K ) Cumulative concentration-response curves for ACh ( F – I ) and SNP ( J and K ) in aorta ( n = 5–8) and basilar artery ( n = 4) ( H – I ). ( L and M ) Cumulative concentration-response curves for 5-HT. ( N ) Mutations in either Cullin-3 or PPARγ cause hypertension. Cullin-3 regulates both the Rho/ROCK pathway through BACURD, whereas Cullin-3 regulates PDE5 through RhoBTB1. RhoBTB1 regulates the activity of PDE5 by ensuring that excess PDE5 is targeted for Cullin-3 dependent ubiquitination and proteasomal degradation. Mutations in PPARγ or treatment with Ang-II causes RhoBTB1-deficiency. All data represent the mean ± SEM. * P

    Article Snippet: To induce expression of RhoBTB1, Tx-free base (MilliporeSigma, T5648) was dissolved in corn oil (MilliporeSigma, C8267) at a concentration of 20 mg/ml by shaking overnight at 37°C.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Mouse Assay, Injection, Concentration Assay, Activity Assay

    BP and vascular function. ( A and B ) Systolic BP was measured by radiotelemetry for 1 week in control ( n = 8), S-P467L ( n = 10), and S-P467L/S-RhoBTB1 mice ( n = 8) before ( A ) or 3 to 4 weeks after Tx treatment ( B ). ( C – E ) Vascular relaxation in control, S-P467L, and S-P467L/S-RhoBTB1 mice after Tx treatment. Cumulative concentration-response curves for ACh ( n = 7–9), or SNP ( n = 7–9) in aorta ( C and D ) and basilar artery ( n = 4–6) ( E ). ( F – H ) Cumulative concentration-response curves for KCl ( n = 8–9) ( F ), ET-1 ( n = 4–6) ( G ), and 5-HT ( n = 4–6) ( H ) in aorta from Tx-treated mice. ( I – L ) Cumulative concentration-response curves for ACh ( n = 4–5) ( I ), SNP ( n = 5–7) ( J ), ET-1 ( n = 4–5) ( K ), and 5-HT ( n = 3) ( L ) in Y-27632–pretreated aorta from the indicated Tx-treated mice. ( M ) Western blot detected p-MYPT, PPARγ, tdTomato, and GAPDH in total aortic protein from the indicated mice after Tx treatment. Shown are 2 representative blots from 6 total samples analyzed for each genotype. Quantification of the p-MYPT results is shown. Data were normalized to the average control value, set to 1.0. All data represent the mean ± SEM. * P

    Journal: The Journal of Clinical Investigation

    Article Title: RhoBTB1 protects against hypertension and arterial stiffness by restraining phosphodiesterase 5 activity

    doi: 10.1172/JCI123462

    Figure Lengend Snippet: BP and vascular function. ( A and B ) Systolic BP was measured by radiotelemetry for 1 week in control ( n = 8), S-P467L ( n = 10), and S-P467L/S-RhoBTB1 mice ( n = 8) before ( A ) or 3 to 4 weeks after Tx treatment ( B ). ( C – E ) Vascular relaxation in control, S-P467L, and S-P467L/S-RhoBTB1 mice after Tx treatment. Cumulative concentration-response curves for ACh ( n = 7–9), or SNP ( n = 7–9) in aorta ( C and D ) and basilar artery ( n = 4–6) ( E ). ( F – H ) Cumulative concentration-response curves for KCl ( n = 8–9) ( F ), ET-1 ( n = 4–6) ( G ), and 5-HT ( n = 4–6) ( H ) in aorta from Tx-treated mice. ( I – L ) Cumulative concentration-response curves for ACh ( n = 4–5) ( I ), SNP ( n = 5–7) ( J ), ET-1 ( n = 4–5) ( K ), and 5-HT ( n = 3) ( L ) in Y-27632–pretreated aorta from the indicated Tx-treated mice. ( M ) Western blot detected p-MYPT, PPARγ, tdTomato, and GAPDH in total aortic protein from the indicated mice after Tx treatment. Shown are 2 representative blots from 6 total samples analyzed for each genotype. Quantification of the p-MYPT results is shown. Data were normalized to the average control value, set to 1.0. All data represent the mean ± SEM. * P

    Article Snippet: To induce expression of RhoBTB1, Tx-free base (MilliporeSigma, T5648) was dissolved in corn oil (MilliporeSigma, C8267) at a concentration of 20 mg/ml by shaking overnight at 37°C.

    Techniques: Mouse Assay, Concentration Assay, Western Blot

    Aortic stiffness. ( A ) Aortic PWV was measured in control, S-P467L, and S-P467L/S-RhoBTB1 mice before Tx treatment or 1, 2, and 3 weeks after completion of the Tx treatment. ( B ) Distensibility was calculated at 3 weeks as described in the Methods. ( C and D ) Compliance curves ( C ) and stress-strain relationships ( D ) were constructed from the inner and outer diameters of aortae from these mice 3–4 weeks after Tx treatment. These parameters were measured at 25-mmHg step changes in pressure from 0 to 200 mmHg. n = 5 for all samples. D, diameter; D 0 , diameter at 0 mmHg. ( E ) Measurement of hydroxyproline as an index of collagen in aorta. ( F ) Perfusion-fixed sections of thoracic aorta 3–4 weeks after Tx treatment were sectioned and stained with Masson’s trichome to stain collagen. Scale bars: 100 μm. The adventitial collagen area was quantified by planimetry. All data represent the mean ± SEM. * P

    Journal: The Journal of Clinical Investigation

    Article Title: RhoBTB1 protects against hypertension and arterial stiffness by restraining phosphodiesterase 5 activity

    doi: 10.1172/JCI123462

    Figure Lengend Snippet: Aortic stiffness. ( A ) Aortic PWV was measured in control, S-P467L, and S-P467L/S-RhoBTB1 mice before Tx treatment or 1, 2, and 3 weeks after completion of the Tx treatment. ( B ) Distensibility was calculated at 3 weeks as described in the Methods. ( C and D ) Compliance curves ( C ) and stress-strain relationships ( D ) were constructed from the inner and outer diameters of aortae from these mice 3–4 weeks after Tx treatment. These parameters were measured at 25-mmHg step changes in pressure from 0 to 200 mmHg. n = 5 for all samples. D, diameter; D 0 , diameter at 0 mmHg. ( E ) Measurement of hydroxyproline as an index of collagen in aorta. ( F ) Perfusion-fixed sections of thoracic aorta 3–4 weeks after Tx treatment were sectioned and stained with Masson’s trichome to stain collagen. Scale bars: 100 μm. The adventitial collagen area was quantified by planimetry. All data represent the mean ± SEM. * P

    Article Snippet: To induce expression of RhoBTB1, Tx-free base (MilliporeSigma, T5648) was dissolved in corn oil (MilliporeSigma, C8267) at a concentration of 20 mg/ml by shaking overnight at 37°C.

    Techniques: Mouse Assay, Construct, Staining

    Molecular mechanism of RhoBTB1 on PDE5. ( A and B ) Reciprocal co-IP of PDE5 and RhoBTB1 in HEK293 cells transfected with Myc-tagged PDE5 and untagged RhoBTB1 ( A ) or His-tagged RhoBTB1 ( B ). Cells in B were treated with MLN4924 (1 μM) for 16 hours. Western blots were probed with the indicated antisera. IP and lysates are labeled. Molecular weight markers were transferred from the original blots. ( C ) Ubiquitination of PDE5 in HEK293 cells transfected with vectors expressing HA-tagged ubiquitin (Ha-Ub) and Cullin-3 in all lanes and, as indicated, Myc-tagged PDE5 and RhoBTB1. Cells were treated with MG132 for the last 2 hours, prior to lysis. The indicated sample was pretreated with MLN4924 (1 μM) for 16 hours. MLN4924 prevents neddylation of Cullin-3 (note the loss of Nedd-C3, the neddylated form of Cullin-3). IP was performed under stringent denaturing conditions to ensure detection of only ubiquitinated PDE5. Under these conditions, RhoBTB1 was not pulled down by PDE5. Western blots were probed with the indicated antisera. IP and lysates are labeled. Molecular weight markers were transferred from the original blots. ( D ) PDE activity in aorta from control mice pretreated with either vehicle (DMSO) or MLN4924 (1 μM) for the indicated durations. ( E ) PDE activity in aorta from control mice pretreated with either MLN4924 (1 μM) or tadalafil (1 μM) for 72 hours. * P

    Journal: The Journal of Clinical Investigation

    Article Title: RhoBTB1 protects against hypertension and arterial stiffness by restraining phosphodiesterase 5 activity

    doi: 10.1172/JCI123462

    Figure Lengend Snippet: Molecular mechanism of RhoBTB1 on PDE5. ( A and B ) Reciprocal co-IP of PDE5 and RhoBTB1 in HEK293 cells transfected with Myc-tagged PDE5 and untagged RhoBTB1 ( A ) or His-tagged RhoBTB1 ( B ). Cells in B were treated with MLN4924 (1 μM) for 16 hours. Western blots were probed with the indicated antisera. IP and lysates are labeled. Molecular weight markers were transferred from the original blots. ( C ) Ubiquitination of PDE5 in HEK293 cells transfected with vectors expressing HA-tagged ubiquitin (Ha-Ub) and Cullin-3 in all lanes and, as indicated, Myc-tagged PDE5 and RhoBTB1. Cells were treated with MG132 for the last 2 hours, prior to lysis. The indicated sample was pretreated with MLN4924 (1 μM) for 16 hours. MLN4924 prevents neddylation of Cullin-3 (note the loss of Nedd-C3, the neddylated form of Cullin-3). IP was performed under stringent denaturing conditions to ensure detection of only ubiquitinated PDE5. Under these conditions, RhoBTB1 was not pulled down by PDE5. Western blots were probed with the indicated antisera. IP and lysates are labeled. Molecular weight markers were transferred from the original blots. ( D ) PDE activity in aorta from control mice pretreated with either vehicle (DMSO) or MLN4924 (1 μM) for the indicated durations. ( E ) PDE activity in aorta from control mice pretreated with either MLN4924 (1 μM) or tadalafil (1 μM) for 72 hours. * P

    Article Snippet: To induce expression of RhoBTB1, Tx-free base (MilliporeSigma, T5648) was dissolved in corn oil (MilliporeSigma, C8267) at a concentration of 20 mg/ml by shaking overnight at 37°C.

    Techniques: Co-Immunoprecipitation Assay, Transfection, Western Blot, Labeling, Molecular Weight, Expressing, Lysis, Activity Assay, Mouse Assay

    Inducible RhoBTB1 experimental model. ( A ) Schematic of the inducible RhoBTB1 transgene construct illustrating removal of the loxP-STOP-loxP with consequent expression of RhoBTB1 and tdTomato in response to Cre recombinase. ( B ) Western blot detecting RhoBTB1 or Myc-tagged RhoBTB1, tdTomato, and GAPDH in HEK293 cells transfected with Myc-RhoBTB1 or RhoBTB1 IND , with or without a Cre recombinase expression vector. Actual size markers transferred from the blots are shown. ( C ) Schematic of breeding to generate triple-transgenic mice expressing dominant-negative PPARγ (S-P467L) in vascular smooth muscle along with inducible smooth muscle–specific expression of RhoBTB1 (S-RhoBTB1). ( D ) Relative mRNA expression of RhoBTB1, tdTomato, and human PPARγ (hPPARγ) was determined by qPCR in aorta from mice of the indicated strains 3–4 weeks after injection of Tx. Data were normalized to the average control value, set to 1.0. All data represent the mean ± SEM. * P

    Journal: The Journal of Clinical Investigation

    Article Title: RhoBTB1 protects against hypertension and arterial stiffness by restraining phosphodiesterase 5 activity

    doi: 10.1172/JCI123462

    Figure Lengend Snippet: Inducible RhoBTB1 experimental model. ( A ) Schematic of the inducible RhoBTB1 transgene construct illustrating removal of the loxP-STOP-loxP with consequent expression of RhoBTB1 and tdTomato in response to Cre recombinase. ( B ) Western blot detecting RhoBTB1 or Myc-tagged RhoBTB1, tdTomato, and GAPDH in HEK293 cells transfected with Myc-RhoBTB1 or RhoBTB1 IND , with or without a Cre recombinase expression vector. Actual size markers transferred from the blots are shown. ( C ) Schematic of breeding to generate triple-transgenic mice expressing dominant-negative PPARγ (S-P467L) in vascular smooth muscle along with inducible smooth muscle–specific expression of RhoBTB1 (S-RhoBTB1). ( D ) Relative mRNA expression of RhoBTB1, tdTomato, and human PPARγ (hPPARγ) was determined by qPCR in aorta from mice of the indicated strains 3–4 weeks after injection of Tx. Data were normalized to the average control value, set to 1.0. All data represent the mean ± SEM. * P

    Article Snippet: To induce expression of RhoBTB1, Tx-free base (MilliporeSigma, T5648) was dissolved in corn oil (MilliporeSigma, C8267) at a concentration of 20 mg/ml by shaking overnight at 37°C.

    Techniques: Construct, Expressing, Western Blot, Transfection, Plasmid Preparation, Transgenic Assay, Mouse Assay, Dominant Negative Mutation, Real-time Polymerase Chain Reaction, Injection

    TAZ deficiency leads to impaired epithelial repair after bleomycin injury. ( A – C ) Lungs of tamoxifen-pretreated SPC-WT-Tm mice were immunostained for TAZ in untreated animals ( A ) or 10 days after bleomycin ( B ). TAZ + tdTomato + traced AEC2s are indicated by yellow arrowheads in ( B ), while other TAZ + cells are indicated by white arrowheads in A . Insets in A also show RAGE costaining. Insets in B show cytoplasmic expression of TAZ in sporadic AEC2s. ( C ) Quantification shows the percentage (mean ± SD) of AEC2 lineage-traced cells expressing TAZ in the nuclei ( n = 3 mice). Total counts: 589 AEC2s (untreated); 916 AEC2s (bleomycin). **** P

    Journal: JCI Insight

    Article Title: TAZ is required for lung alveolar epithelial cell differentiation after injury

    doi: 10.1172/jci.insight.128674

    Figure Lengend Snippet: TAZ deficiency leads to impaired epithelial repair after bleomycin injury. ( A – C ) Lungs of tamoxifen-pretreated SPC-WT-Tm mice were immunostained for TAZ in untreated animals ( A ) or 10 days after bleomycin ( B ). TAZ + tdTomato + traced AEC2s are indicated by yellow arrowheads in ( B ), while other TAZ + cells are indicated by white arrowheads in A . Insets in A also show RAGE costaining. Insets in B show cytoplasmic expression of TAZ in sporadic AEC2s. ( C ) Quantification shows the percentage (mean ± SD) of AEC2 lineage-traced cells expressing TAZ in the nuclei ( n = 3 mice). Total counts: 589 AEC2s (untreated); 916 AEC2s (bleomycin). **** P

    Article Snippet: Induction of CreERT2 alleles was done by i.p. injecting 80 mg/kg tamoxifen (T5648; MilliporeSigma) in sunflower seed oil (S5007-1L; MilliporeSigma) for 5 consecutive days.

    Techniques: Mouse Assay, Expressing

    IWR-1 inhibits AEC2-to-AEC1 differentiation in vitro. TdTomato + lineage-labeled AEC2s were sorted from SPC-CreERT2; Rosa-lsl-tdTomato mouse lungs after tamoxifen administration. ( A ) 3D alveolar spheroid culture schematic: AEC2s were cultured in Matrigel with NHLF for 2 weeks or until alveolospheres developed. ( B – G ) Cells were treated with either control (DMSO) ( B – D ) or IWR-1 ( E – G ) throughout culture. Merged bright-field (BF) and fluorescence (FL) full-plate images ( B and E ) and FL close-ups ( C and F ) show the overall difference in shapes of the cultured spheroids under these 2 conditions. ( D and G ) Representative high-power BF images emphasize the distinct smooth surface of spheroids cultured with IWR-1 as compared to controls. ( H and I) Whole spheroids were immunostained for AEC2 (SPC), AEC1 (HOPX), and Nuclei (Hoechst). Single focal planes of Z -stack images are shown. Full Z (control) and 2 (IWR-1). ( J ) Gene expression ratios (mean ± SD, 3 biological replicates) between Spc and Hopx , Aqp5 , and Pdpn were used to quantify the AEC2 versus AEC1 composition of each well. No signals were detected with NHLF alone with these mouse qPCR probes. * P

    Journal: JCI Insight

    Article Title: TAZ is required for lung alveolar epithelial cell differentiation after injury

    doi: 10.1172/jci.insight.128674

    Figure Lengend Snippet: IWR-1 inhibits AEC2-to-AEC1 differentiation in vitro. TdTomato + lineage-labeled AEC2s were sorted from SPC-CreERT2; Rosa-lsl-tdTomato mouse lungs after tamoxifen administration. ( A ) 3D alveolar spheroid culture schematic: AEC2s were cultured in Matrigel with NHLF for 2 weeks or until alveolospheres developed. ( B – G ) Cells were treated with either control (DMSO) ( B – D ) or IWR-1 ( E – G ) throughout culture. Merged bright-field (BF) and fluorescence (FL) full-plate images ( B and E ) and FL close-ups ( C and F ) show the overall difference in shapes of the cultured spheroids under these 2 conditions. ( D and G ) Representative high-power BF images emphasize the distinct smooth surface of spheroids cultured with IWR-1 as compared to controls. ( H and I) Whole spheroids were immunostained for AEC2 (SPC), AEC1 (HOPX), and Nuclei (Hoechst). Single focal planes of Z -stack images are shown. Full Z (control) and 2 (IWR-1). ( J ) Gene expression ratios (mean ± SD, 3 biological replicates) between Spc and Hopx , Aqp5 , and Pdpn were used to quantify the AEC2 versus AEC1 composition of each well. No signals were detected with NHLF alone with these mouse qPCR probes. * P

    Article Snippet: Induction of CreERT2 alleles was done by i.p. injecting 80 mg/kg tamoxifen (T5648; MilliporeSigma) in sunflower seed oil (S5007-1L; MilliporeSigma) for 5 consecutive days.

    Techniques: In Vitro, Labeling, Cell Culture, Fluorescence, Expressing, Real-time Polymerase Chain Reaction