att 20 cell line  (ATCC)


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    Structured Review

    ATCC att 20 cell line
    Cell-specific chromatin landscapes determine cell-selective GR occupancy (a–b) Pituitary-specific GR occupancy dictated by pituitary-specific DNaseI sensitivity transitions. Shown are examples of DNaseI sensitivity and GR occupancy patterns in relation to hormone exposure comparing mouse mammary (3134) and pituitary <t>(AtT-20)</t> cells (see Fig.1 legend and Supplementary Fig.8a-c for additional examples). (c) Global GR occupancy vs. chromatin accessibility landscape in pituitary cells. In pituitary cells, virtually all sites of GR occupancy (94.9%, 3,079/3,242 sites) occur within pre-hormone accessible chromatin. The small fraction of re-progra mmed GR sites (138 GR ChIP peaks, 4.2% of total) is shown in red. As in mammary cells, only a small fraction of pre-hormone accessible chromatin is occupied (note: for legibility, GR circle shown at 5X scale). (d) Significant differences in genomic distribution of pre-hormone DNaseI sensitivity in mammary (grey) vs. pituitary (green) cells; only 0.78% of genome (20.5Mb) is accessible in both cell types. (e) GR occupancy is highly cell-selective. Only 371 GR occupancy sites are shared between mammary and pituitary cells (4.5% of 3134 sites and 11.4% of AtT-20 sites).
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    Images

    1) Product Images from "Chromatin accessibility pre-determines glucocorticoid receptor binding patterns"

    Article Title: Chromatin accessibility pre-determines glucocorticoid receptor binding patterns

    Journal: Nature genetics

    doi: 10.1038/ng.759

    Cell-specific chromatin landscapes determine cell-selective GR occupancy (a–b) Pituitary-specific GR occupancy dictated by pituitary-specific DNaseI sensitivity transitions. Shown are examples of DNaseI sensitivity and GR occupancy patterns in relation to hormone exposure comparing mouse mammary (3134) and pituitary (AtT-20) cells (see Fig.1 legend and Supplementary Fig.8a-c for additional examples). (c) Global GR occupancy vs. chromatin accessibility landscape in pituitary cells. In pituitary cells, virtually all sites of GR occupancy (94.9%, 3,079/3,242 sites) occur within pre-hormone accessible chromatin. The small fraction of re-progra mmed GR sites (138 GR ChIP peaks, 4.2% of total) is shown in red. As in mammary cells, only a small fraction of pre-hormone accessible chromatin is occupied (note: for legibility, GR circle shown at 5X scale). (d) Significant differences in genomic distribution of pre-hormone DNaseI sensitivity in mammary (grey) vs. pituitary (green) cells; only 0.78% of genome (20.5Mb) is accessible in both cell types. (e) GR occupancy is highly cell-selective. Only 371 GR occupancy sites are shared between mammary and pituitary cells (4.5% of 3134 sites and 11.4% of AtT-20 sites).
    Figure Legend Snippet: Cell-specific chromatin landscapes determine cell-selective GR occupancy (a–b) Pituitary-specific GR occupancy dictated by pituitary-specific DNaseI sensitivity transitions. Shown are examples of DNaseI sensitivity and GR occupancy patterns in relation to hormone exposure comparing mouse mammary (3134) and pituitary (AtT-20) cells (see Fig.1 legend and Supplementary Fig.8a-c for additional examples). (c) Global GR occupancy vs. chromatin accessibility landscape in pituitary cells. In pituitary cells, virtually all sites of GR occupancy (94.9%, 3,079/3,242 sites) occur within pre-hormone accessible chromatin. The small fraction of re-progra mmed GR sites (138 GR ChIP peaks, 4.2% of total) is shown in red. As in mammary cells, only a small fraction of pre-hormone accessible chromatin is occupied (note: for legibility, GR circle shown at 5X scale). (d) Significant differences in genomic distribution of pre-hormone DNaseI sensitivity in mammary (grey) vs. pituitary (green) cells; only 0.78% of genome (20.5Mb) is accessible in both cell types. (e) GR occupancy is highly cell-selective. Only 371 GR occupancy sites are shared between mammary and pituitary cells (4.5% of 3134 sites and 11.4% of AtT-20 sites).

    Techniques Used: Chromatin Immunoprecipitation

    Regulatory motifs in GR-occupied regions differ substantially between cell types (a–b) Results of de novo motif discovery (see Supplementary Notes ) performed on the top 500 GR occupancy sites identified in 3134 (panel a ) and AtT-20 (panel b ). The GR sites were further separated into pre-programmed (GR occupancy within pre-hormone accessible chromatin) vs. re-programmed (GR occupancy within pre-hormone inaccessible chromatin) sites. Shown are motifs with highly significant enrichment (e
    Figure Legend Snippet: Regulatory motifs in GR-occupied regions differ substantially between cell types (a–b) Results of de novo motif discovery (see Supplementary Notes ) performed on the top 500 GR occupancy sites identified in 3134 (panel a ) and AtT-20 (panel b ). The GR sites were further separated into pre-programmed (GR occupancy within pre-hormone accessible chromatin) vs. re-programmed (GR occupancy within pre-hormone inaccessible chromatin) sites. Shown are motifs with highly significant enrichment (e

    Techniques Used:

    2) Product Images from "Cell Cycle-Dependent Localization of Voltage-Dependent Calcium Channels and the Mitotic Apparatus in a Neuroendocrine Cell Line(AtT-20)"

    Article Title: Cell Cycle-Dependent Localization of Voltage-Dependent Calcium Channels and the Mitotic Apparatus in a Neuroendocrine Cell Line(AtT-20)

    Journal: International Journal of Cell Biology

    doi: 10.1155/2009/487959

    Sites of calcium transients during mitosis . Calcium transients correspond with sites of CaV1 immunofluorescence in AtT-20 Cells during mitosis. Simultaneous imaging of (a) Fluo4 (4 μ M) intracellular calcium transients and (b) cell morphology by DIC in AtT-20 cells during cytokinesis. Regions: 1=midbody; 2, 3=spindle mid-zone; 4=nuclear region of interphase cell; 5=background (media only). (c) Plot of fluorescence units for each region over time (sec). Regions of enhanced calcium transients correspond to midbody (1) and spindle mid-zone (2, 3). Break in X-axis marks time of acquisition following loading of Fluo-4.
    Figure Legend Snippet: Sites of calcium transients during mitosis . Calcium transients correspond with sites of CaV1 immunofluorescence in AtT-20 Cells during mitosis. Simultaneous imaging of (a) Fluo4 (4 μ M) intracellular calcium transients and (b) cell morphology by DIC in AtT-20 cells during cytokinesis. Regions: 1=midbody; 2, 3=spindle mid-zone; 4=nuclear region of interphase cell; 5=background (media only). (c) Plot of fluorescence units for each region over time (sec). Regions of enhanced calcium transients correspond to midbody (1) and spindle mid-zone (2, 3). Break in X-axis marks time of acquisition following loading of Fluo-4.

    Techniques Used: Immunofluorescence, Imaging, Fluorescence

    Calcium channel CaV1.2 and CaV1.3 localization in mitotic cells . 1(A) compares CaV1.2 staining in AtT-20 cells (a)–(c), PC-12 cells (d), and INS-1 cells (e)–(g). CaV1.2 is located with kinetechores at the “poleward side” during metaphase and anaphase ((a), (b): inset) in the AtT-20 cells. Midbody CaV1.2 staining is observed in both AtT-20 cells and PC-12 cells (c), (d) but not in INS-1 cells (g). 1(B) is a similar comparison for CaV1.3 and shows staining at the mid-spindle zone only in AtT-20s during telophase (a). In contrast, all three neuroendocrine cell types display CaV1.3 staining at the midbody during cytokinesis ((B): (b), (c), and (e)). 1(C) shows control staining of Cos 7 cells; in particular, note that there is no nonspecific staining at the midbody of these cells (1(C) (c), (e)). CaV1.2 and CaV1.3 are stained with antibodies specific to alpha subunit sequences for CaV1.2 and CaV1.3 (see Methods, Alamone Labs) and detected with secondary goat anti-rabbit antibody Alexa 555 (red); Microtubules are stained with DM1A and goat anti-mouse Alexa 647 (blue); kinetechores are stained with anti-CREST antibody and Alexa 488 (green). Bleedthrough between microtubule and CREST staining channels was corrected for as described in methods.
    Figure Legend Snippet: Calcium channel CaV1.2 and CaV1.3 localization in mitotic cells . 1(A) compares CaV1.2 staining in AtT-20 cells (a)–(c), PC-12 cells (d), and INS-1 cells (e)–(g). CaV1.2 is located with kinetechores at the “poleward side” during metaphase and anaphase ((a), (b): inset) in the AtT-20 cells. Midbody CaV1.2 staining is observed in both AtT-20 cells and PC-12 cells (c), (d) but not in INS-1 cells (g). 1(B) is a similar comparison for CaV1.3 and shows staining at the mid-spindle zone only in AtT-20s during telophase (a). In contrast, all three neuroendocrine cell types display CaV1.3 staining at the midbody during cytokinesis ((B): (b), (c), and (e)). 1(C) shows control staining of Cos 7 cells; in particular, note that there is no nonspecific staining at the midbody of these cells (1(C) (c), (e)). CaV1.2 and CaV1.3 are stained with antibodies specific to alpha subunit sequences for CaV1.2 and CaV1.3 (see Methods, Alamone Labs) and detected with secondary goat anti-rabbit antibody Alexa 555 (red); Microtubules are stained with DM1A and goat anti-mouse Alexa 647 (blue); kinetechores are stained with anti-CREST antibody and Alexa 488 (green). Bleedthrough between microtubule and CREST staining channels was corrected for as described in methods.

    Techniques Used: Staining

    Nifedipine attenuates AtT-20 Cell density . (a) Cell viability . Incubation with the DHP CaV antagonist, nifedipine, results in a dose-dependent decrease in AtT-20 cell number using the colorimetric MTS assay for cell viability. The half maximal dose for this effect is 1.5 ± 0.3 μ M nifedipine. Graph shows normalized data from 3 experiments. (b) Cell proliferation and mitotic stages. Graph shows mean number of cells ±SE for at least 5 fields (≥ 800 total cells counted for Day 3 for each condition). There is a significant decrease in the total number of cells counted between vehicle control and nifedipine-treated cells (* P
    Figure Legend Snippet: Nifedipine attenuates AtT-20 Cell density . (a) Cell viability . Incubation with the DHP CaV antagonist, nifedipine, results in a dose-dependent decrease in AtT-20 cell number using the colorimetric MTS assay for cell viability. The half maximal dose for this effect is 1.5 ± 0.3 μ M nifedipine. Graph shows normalized data from 3 experiments. (b) Cell proliferation and mitotic stages. Graph shows mean number of cells ±SE for at least 5 fields (≥ 800 total cells counted for Day 3 for each condition). There is a significant decrease in the total number of cells counted between vehicle control and nifedipine-treated cells (* P

    Techniques Used: Incubation, MTS Assay

    3) Product Images from "Region-Specific Regulation of RGS4 (Regulator of G-Protein–Signaling Protein Type 4) in Brain by Stress and Glucocorticoids: In Vivo and In VitroStudies"

    Article Title: Region-Specific Regulation of RGS4 (Regulator of G-Protein–Signaling Protein Type 4) in Brain by Stress and Glucocorticoids: In Vivo and In VitroStudies

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.19-10-03674.1999

    Effect of actinomycin D on dexamethasone regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 4).
    Figure Legend Snippet: Effect of actinomycin D on dexamethasone regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 4).

    Techniques Used: Expressing

    Time course study for dexamethasone regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 3–6; * p
    Figure Legend Snippet: Time course study for dexamethasone regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 3–6; * p

    Techniques Used: Expressing

    Dose–response analyses for dexamethasone ( Dex ) regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 3–6; * p
    Figure Legend Snippet: Dose–response analyses for dexamethasone ( Dex ) regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 3–6; * p

    Techniques Used: Expressing

    Comparison of the regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells by dexamethasone ( Dex ), CRF, and forskolin ( FK ). Data are expressed as the mean percent of control (± SEM; n = 4; * p
    Figure Legend Snippet: Comparison of the regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells by dexamethasone ( Dex ), CRF, and forskolin ( FK ). Data are expressed as the mean percent of control (± SEM; n = 4; * p

    Techniques Used: Expressing

    4) Product Images from "Region-Specific Regulation of RGS4 (Regulator of G-Protein–Signaling Protein Type 4) in Brain by Stress and Glucocorticoids: In Vivo and In VitroStudies"

    Article Title: Region-Specific Regulation of RGS4 (Regulator of G-Protein–Signaling Protein Type 4) in Brain by Stress and Glucocorticoids: In Vivo and In VitroStudies

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.19-10-03674.1999

    Effect of actinomycin D on dexamethasone regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 4).
    Figure Legend Snippet: Effect of actinomycin D on dexamethasone regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 4).

    Techniques Used: Expressing

    Time course study for dexamethasone regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 3–6; * p
    Figure Legend Snippet: Time course study for dexamethasone regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 3–6; * p

    Techniques Used: Expressing

    Dose–response analyses for dexamethasone ( Dex ) regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 3–6; * p
    Figure Legend Snippet: Dose–response analyses for dexamethasone ( Dex ) regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 3–6; * p

    Techniques Used: Expressing

    Comparison of the regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells by dexamethasone ( Dex ), CRF, and forskolin ( FK ). Data are expressed as the mean percent of control (± SEM; n = 4; * p
    Figure Legend Snippet: Comparison of the regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells by dexamethasone ( Dex ), CRF, and forskolin ( FK ). Data are expressed as the mean percent of control (± SEM; n = 4; * p

    Techniques Used: Expressing

    5) Product Images from "A Novel Mechanism Regulating Dopamine Receptor Type 2 Signal Transduction in Pituitary Tumoral Cells: The Role of cAMP/PKA-Induced Filamin A Phosphorylation"

    Article Title: A Novel Mechanism Regulating Dopamine Receptor Type 2 Signal Transduction in Pituitary Tumoral Cells: The Role of cAMP/PKA-Induced Filamin A Phosphorylation

    Journal: Frontiers in Endocrinology

    doi: 10.3389/fendo.2020.611752

    FLNA S2152D mutant reverted DRD2 inhibitory effects on cell proliferation in both MMQ and AtT-20 cells. MMQ (A, C, E) and AtT-20 cells (B, D, F) were transiently transfected with empty vector, wild-type, S2152A and S2152D FLNA mutants for 72 h at 37°C. MMQ (A) and AtT-20 (B) were than incubated 72 h or 96 h with 100 nM BIM53097, respectively. BrdU incorporation in newly synthesized DNA was measured. Experiments were repeated 4 times and each determination was done in quadruplicate. *p
    Figure Legend Snippet: FLNA S2152D mutant reverted DRD2 inhibitory effects on cell proliferation in both MMQ and AtT-20 cells. MMQ (A, C, E) and AtT-20 cells (B, D, F) were transiently transfected with empty vector, wild-type, S2152A and S2152D FLNA mutants for 72 h at 37°C. MMQ (A) and AtT-20 (B) were than incubated 72 h or 96 h with 100 nM BIM53097, respectively. BrdU incorporation in newly synthesized DNA was measured. Experiments were repeated 4 times and each determination was done in quadruplicate. *p

    Techniques Used: Mutagenesis, Transfection, Plasmid Preparation, Incubation, BrdU Incorporation Assay, Synthesized

    FLNA phosphorylation is reduced by DRD2 agonist and increased by forskolin treatment, respectively. MMQ cells (A, C) and AtT-20 cells (B, D) were treated with 1μM forskolin or 100 nM BIM53097 for 10 min (A, B) or indicated times (C, D) at 37°C. The graphs show the quantification of P-FLNA/total FLNA ratio. Experiments were repeated 5 times. Values represent mean ± S.D. normalized vs. respective basal. Representative immunoblots are shown. *p
    Figure Legend Snippet: FLNA phosphorylation is reduced by DRD2 agonist and increased by forskolin treatment, respectively. MMQ cells (A, C) and AtT-20 cells (B, D) were treated with 1μM forskolin or 100 nM BIM53097 for 10 min (A, B) or indicated times (C, D) at 37°C. The graphs show the quantification of P-FLNA/total FLNA ratio. Experiments were repeated 5 times. Values represent mean ± S.D. normalized vs. respective basal. Representative immunoblots are shown. *p

    Techniques Used: Western Blot

    Effects of S2152D FLNA mutant overexpression on ERK1/2 and AKT phosphorylation. MMQ (A, C) and AtT-20 cells (B) were treated 10 min with 100 nM BIM53097. The graphs show the quantification of P-ERK1/2 expression levels normalized to total ERK1/2 ( A and B ) and P-AKT/total AKT (C) . Data represent mean ± S.D. from three independent experiments. Representative immunoblots are shown. *p
    Figure Legend Snippet: Effects of S2152D FLNA mutant overexpression on ERK1/2 and AKT phosphorylation. MMQ (A, C) and AtT-20 cells (B) were treated 10 min with 100 nM BIM53097. The graphs show the quantification of P-ERK1/2 expression levels normalized to total ERK1/2 ( A and B ) and P-AKT/total AKT (C) . Data represent mean ± S.D. from three independent experiments. Representative immunoblots are shown. *p

    Techniques Used: Mutagenesis, Over Expression, Expressing, Western Blot

    FLNA phosphorylation abolished antisecretory effects of DRD2. MMQ (A) and AtT-20 cells (B) were transiently transfected with wild-type, S2152A and S2152D FLNA mutants for 72 h at 37°C. After 4 h (MMQ) or 24 h (AtT-20) BIM53097 treatment, hormonal assay was performed in order to detect hormones levels. The graphs show the percentage of PRL (A) and ACTH (B) secretion levels. Data represent mean ± S.D. from three independent experiments and each determination was done in triplicate. *p
    Figure Legend Snippet: FLNA phosphorylation abolished antisecretory effects of DRD2. MMQ (A) and AtT-20 cells (B) were transiently transfected with wild-type, S2152A and S2152D FLNA mutants for 72 h at 37°C. After 4 h (MMQ) or 24 h (AtT-20) BIM53097 treatment, hormonal assay was performed in order to detect hormones levels. The graphs show the percentage of PRL (A) and ACTH (B) secretion levels. Data represent mean ± S.D. from three independent experiments and each determination was done in triplicate. *p

    Techniques Used: Transfection, Hormonal Assay

    6) Product Images from "MicroRNA-30d target TIMP3 induces pituitary tumor cell growth and invasion"

    Article Title: MicroRNA-30d target TIMP3 induces pituitary tumor cell growth and invasion

    Journal: Gland Surgery

    doi: 10.21037/gs-21-720

    MiR-30d could promote AtT-20 cell proliferation and invasion via TIMP3. (A) Detection of the interference effects of different TIMP3 siRNAs (*, P
    Figure Legend Snippet: MiR-30d could promote AtT-20 cell proliferation and invasion via TIMP3. (A) Detection of the interference effects of different TIMP3 siRNAs (*, P

    Techniques Used:

    Silencing of miR-30d could inhibit AtT-20 cell proliferation and invasion. (A,B) qRT-PCR was used to determine the expression of miR-30d after transfection with miR-30d mimics or inhibitor (n=3, *, P
    Figure Legend Snippet: Silencing of miR-30d could inhibit AtT-20 cell proliferation and invasion. (A,B) qRT-PCR was used to determine the expression of miR-30d after transfection with miR-30d mimics or inhibitor (n=3, *, P

    Techniques Used: Quantitative RT-PCR, Expressing, Transfection

    7) Product Images from "Region-Specific Regulation of RGS4 (Regulator of G-Protein–Signaling Protein Type 4) in Brain by Stress and Glucocorticoids: In Vivo and In VitroStudies"

    Article Title: Region-Specific Regulation of RGS4 (Regulator of G-Protein–Signaling Protein Type 4) in Brain by Stress and Glucocorticoids: In Vivo and In VitroStudies

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.19-10-03674.1999

    Effect of actinomycin D on dexamethasone regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 4).
    Figure Legend Snippet: Effect of actinomycin D on dexamethasone regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 4).

    Techniques Used: Expressing

    Time course study for dexamethasone regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 3–6; * p
    Figure Legend Snippet: Time course study for dexamethasone regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 3–6; * p

    Techniques Used: Expressing

    Dose–response analyses for dexamethasone ( Dex ) regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 3–6; * p
    Figure Legend Snippet: Dose–response analyses for dexamethasone ( Dex ) regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 3–6; * p

    Techniques Used: Expressing

    Comparison of the regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells by dexamethasone ( Dex ), CRF, and forskolin ( FK ). Data are expressed as the mean percent of control (± SEM; n = 4; * p
    Figure Legend Snippet: Comparison of the regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells by dexamethasone ( Dex ), CRF, and forskolin ( FK ). Data are expressed as the mean percent of control (± SEM; n = 4; * p

    Techniques Used: Expressing

    8) Product Images from "Identification of driver genes and key pathways of non-functional pituitary adenomas predicts the therapeutic effect of STO-609"

    Article Title: Identification of driver genes and key pathways of non-functional pituitary adenomas predicts the therapeutic effect of STO-609

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0240230

    (A) Cellular viability of HP75, GT1-1 and AtT-20 cells treated with STO-609 (B) Clonogenicities in Petri dishes with different doses of STO-609 (C) Scratch assay in control and STO-609 group (D) numbers of clone formation in HP75 cell line and GT1-1 cell line (E) Wound width in control and STO-609 (F) The distribution of cells in apoptosis with different doses of STO-609 (G) The percentage of apoptosis cell treated with different dose of STO-609.
    Figure Legend Snippet: (A) Cellular viability of HP75, GT1-1 and AtT-20 cells treated with STO-609 (B) Clonogenicities in Petri dishes with different doses of STO-609 (C) Scratch assay in control and STO-609 group (D) numbers of clone formation in HP75 cell line and GT1-1 cell line (E) Wound width in control and STO-609 (F) The distribution of cells in apoptosis with different doses of STO-609 (G) The percentage of apoptosis cell treated with different dose of STO-609.

    Techniques Used: Wound Healing Assay

    9) Product Images from "Glucocorticoid-induced loss of DNA methylation in non-neuronal cells and potential involvement of DNMT1 in epigenetic regulation of Fkbp5"

    Article Title: Glucocorticoid-induced loss of DNA methylation in non-neuronal cells and potential involvement of DNMT1 in epigenetic regulation of Fkbp5

    Journal: Biochemical and Biophysical Research Communications

    doi: 10.1016/j.bbrc.2012.03.035

    Glucocorticoid-induced increase in Fkbp5 gene expression and decrease in DNAm in the AtT-20 cell line. (A) Fkbp5 gene expression increases with dexamethasone (DEX) treatment in a dose-dependent manner in the AtT-20 cell line. (B) Increase in gene expression
    Figure Legend Snippet: Glucocorticoid-induced increase in Fkbp5 gene expression and decrease in DNAm in the AtT-20 cell line. (A) Fkbp5 gene expression increases with dexamethasone (DEX) treatment in a dose-dependent manner in the AtT-20 cell line. (B) Increase in gene expression

    Techniques Used: Expressing

    10) Product Images from "DNA methylation status predicts cell type-specific enhancer activity"

    Article Title: DNA methylation status predicts cell type-specific enhancer activity

    Journal: The EMBO Journal

    doi: 10.1038/emboj.2011.210

    Cytosine demethylation marks the regions of chromatin transition and correlates with GR binding in a cell type-specific manner. DNA was extracted from untreated 3134 and AtT-20 cells and analysed by bisulphite sequencing. The following pre-programmed
    Figure Legend Snippet: Cytosine demethylation marks the regions of chromatin transition and correlates with GR binding in a cell type-specific manner. DNA was extracted from untreated 3134 and AtT-20 cells and analysed by bisulphite sequencing. The following pre-programmed

    Techniques Used: Binding Assay, Bisulfite Sequencing

    11) Product Images from "Erythropoietin negatively regulates pituitary ACTH secretion"

    Article Title: Erythropoietin negatively regulates pituitary ACTH secretion

    Journal: Brain research

    doi: 10.1016/j.brainres.2015.02.052

    Epo regulates ACTH secretion from the murine pituitary cell line AtT-20
    Figure Legend Snippet: Epo regulates ACTH secretion from the murine pituitary cell line AtT-20

    Techniques Used:

    12) Product Images from "miR-15a/miR-16-1 expression inversely correlates with cyclin D1 levels in Men1 pituitary NETs"

    Article Title: miR-15a/miR-16-1 expression inversely correlates with cyclin D1 levels in Men1 pituitary NETs

    Journal: The Journal of Endocrinology

    doi: 10.1530/JOE-18-0278

    Relationship between miR-15a/miR-16-1 and menin in HeLa and AtT20 cells. Significant differences were not observed in the levels of Men1 or menin expression after antagomir treatment of HeLa cells, evaluated by qRT-PCR (A) and Western blot (B), respectively. Use of MEN1 -specific siRNA in HeLa and AtT20 cells decreased expression of MEN1 (C) (assessed by qRT-PCR, *** P
    Figure Legend Snippet: Relationship between miR-15a/miR-16-1 and menin in HeLa and AtT20 cells. Significant differences were not observed in the levels of Men1 or menin expression after antagomir treatment of HeLa cells, evaluated by qRT-PCR (A) and Western blot (B), respectively. Use of MEN1 -specific siRNA in HeLa and AtT20 cells decreased expression of MEN1 (C) (assessed by qRT-PCR, *** P

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot

    13) Product Images from "A Novel Mechanism Regulating Dopamine Receptor Type 2 Signal Transduction in Pituitary Tumoral Cells: The Role of cAMP/PKA-Induced Filamin A Phosphorylation"

    Article Title: A Novel Mechanism Regulating Dopamine Receptor Type 2 Signal Transduction in Pituitary Tumoral Cells: The Role of cAMP/PKA-Induced Filamin A Phosphorylation

    Journal: Frontiers in Endocrinology

    doi: 10.3389/fendo.2020.611752

    FLNA S2152D mutant reverted DRD2 inhibitory effects on cell proliferation in both MMQ and AtT-20 cells. MMQ (A, C, E) and AtT-20 cells (B, D, F) were transiently transfected with empty vector, wild-type, S2152A and S2152D FLNA mutants for 72 h at 37°C. MMQ (A) and AtT-20 (B) were than incubated 72 h or 96 h with 100 nM BIM53097, respectively. BrdU incorporation in newly synthesized DNA was measured. Experiments were repeated 4 times and each determination was done in quadruplicate. *p
    Figure Legend Snippet: FLNA S2152D mutant reverted DRD2 inhibitory effects on cell proliferation in both MMQ and AtT-20 cells. MMQ (A, C, E) and AtT-20 cells (B, D, F) were transiently transfected with empty vector, wild-type, S2152A and S2152D FLNA mutants for 72 h at 37°C. MMQ (A) and AtT-20 (B) were than incubated 72 h or 96 h with 100 nM BIM53097, respectively. BrdU incorporation in newly synthesized DNA was measured. Experiments were repeated 4 times and each determination was done in quadruplicate. *p

    Techniques Used: Mutagenesis, Transfection, Plasmid Preparation, Incubation, BrdU Incorporation Assay, Synthesized

    FLNA phosphorylation is reduced by DRD2 agonist and increased by forskolin treatment, respectively. MMQ cells (A, C) and AtT-20 cells (B, D) were treated with 1μM forskolin or 100 nM BIM53097 for 10 min (A, B) or indicated times (C, D) at 37°C. The graphs show the quantification of P-FLNA/total FLNA ratio. Experiments were repeated 5 times. Values represent mean ± S.D. normalized vs. respective basal. Representative immunoblots are shown. *p
    Figure Legend Snippet: FLNA phosphorylation is reduced by DRD2 agonist and increased by forskolin treatment, respectively. MMQ cells (A, C) and AtT-20 cells (B, D) were treated with 1μM forskolin or 100 nM BIM53097 for 10 min (A, B) or indicated times (C, D) at 37°C. The graphs show the quantification of P-FLNA/total FLNA ratio. Experiments were repeated 5 times. Values represent mean ± S.D. normalized vs. respective basal. Representative immunoblots are shown. *p

    Techniques Used: Western Blot

    Effects of S2152D FLNA mutant overexpression on ERK1/2 and AKT phosphorylation. MMQ (A, C) and AtT-20 cells (B) were treated 10 min with 100 nM BIM53097. The graphs show the quantification of P-ERK1/2 expression levels normalized to total ERK1/2 ( A and B ) and P-AKT/total AKT (C) . Data represent mean ± S.D. from three independent experiments. Representative immunoblots are shown. *p
    Figure Legend Snippet: Effects of S2152D FLNA mutant overexpression on ERK1/2 and AKT phosphorylation. MMQ (A, C) and AtT-20 cells (B) were treated 10 min with 100 nM BIM53097. The graphs show the quantification of P-ERK1/2 expression levels normalized to total ERK1/2 ( A and B ) and P-AKT/total AKT (C) . Data represent mean ± S.D. from three independent experiments. Representative immunoblots are shown. *p

    Techniques Used: Mutagenesis, Over Expression, Expressing, Western Blot

    FLNA phosphorylation abolished antisecretory effects of DRD2. MMQ (A) and AtT-20 cells (B) were transiently transfected with wild-type, S2152A and S2152D FLNA mutants for 72 h at 37°C. After 4 h (MMQ) or 24 h (AtT-20) BIM53097 treatment, hormonal assay was performed in order to detect hormones levels. The graphs show the percentage of PRL (A) and ACTH (B) secretion levels. Data represent mean ± S.D. from three independent experiments and each determination was done in triplicate. *p
    Figure Legend Snippet: FLNA phosphorylation abolished antisecretory effects of DRD2. MMQ (A) and AtT-20 cells (B) were transiently transfected with wild-type, S2152A and S2152D FLNA mutants for 72 h at 37°C. After 4 h (MMQ) or 24 h (AtT-20) BIM53097 treatment, hormonal assay was performed in order to detect hormones levels. The graphs show the percentage of PRL (A) and ACTH (B) secretion levels. Data represent mean ± S.D. from three independent experiments and each determination was done in triplicate. *p

    Techniques Used: Transfection, Hormonal Assay

    14) Product Images from "Glucocorticoid Receptor Antagonism Upregulates Somatostatin Receptor Subtype 2 Expression in ACTH-Producing Neuroendocrine Tumors: New Insight Based on the Selective Glucocorticoid Receptor Modulator Relacorilant"

    Article Title: Glucocorticoid Receptor Antagonism Upregulates Somatostatin Receptor Subtype 2 Expression in ACTH-Producing Neuroendocrine Tumors: New Insight Based on the Selective Glucocorticoid Receptor Modulator Relacorilant

    Journal: Frontiers in Endocrinology

    doi: 10.3389/fendo.2021.793262

    Log 2 fold change in SSTR2 mRNA in murine At-T20 cells upon treatment with increasing concentrations of relacorilant for 24 h in the presence of 100 nM dexamethasone. 0%, 2-fold, and 3-fold inhibition and 1.5-fold increase in levels are highlighted by dotted lines on the y-axis. Zero relative expression is in the absence of dexamethasone. Data points show average fold change compared to baseline and SD error bars. Data are technical replicates with an underlying n=1.
    Figure Legend Snippet: Log 2 fold change in SSTR2 mRNA in murine At-T20 cells upon treatment with increasing concentrations of relacorilant for 24 h in the presence of 100 nM dexamethasone. 0%, 2-fold, and 3-fold inhibition and 1.5-fold increase in levels are highlighted by dotted lines on the y-axis. Zero relative expression is in the absence of dexamethasone. Data points show average fold change compared to baseline and SD error bars. Data are technical replicates with an underlying n=1.

    Techniques Used: Inhibition, Expressing

    15) Product Images from "Region-Specific Regulation of RGS4 (Regulator of G-Protein–Signaling Protein Type 4) in Brain by Stress and Glucocorticoids: In Vivo and In VitroStudies"

    Article Title: Region-Specific Regulation of RGS4 (Regulator of G-Protein–Signaling Protein Type 4) in Brain by Stress and Glucocorticoids: In Vivo and In VitroStudies

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.19-10-03674.1999

    Effect of actinomycin D on dexamethasone regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 4).
    Figure Legend Snippet: Effect of actinomycin D on dexamethasone regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 4).

    Techniques Used: Expressing

    Time course study for dexamethasone regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 3–6; * p
    Figure Legend Snippet: Time course study for dexamethasone regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 3–6; * p

    Techniques Used: Expressing

    Dose–response analyses for dexamethasone ( Dex ) regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 3–6; * p
    Figure Legend Snippet: Dose–response analyses for dexamethasone ( Dex ) regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 3–6; * p

    Techniques Used: Expressing

    Comparison of the regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells by dexamethasone ( Dex ), CRF, and forskolin ( FK ). Data are expressed as the mean percent of control (± SEM; n = 4; * p
    Figure Legend Snippet: Comparison of the regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells by dexamethasone ( Dex ), CRF, and forskolin ( FK ). Data are expressed as the mean percent of control (± SEM; n = 4; * p

    Techniques Used: Expressing

    16) Product Images from "Evidence for orphan nuclear receptor TR4 in the etiology of Cushing disease"

    Article Title: Evidence for orphan nuclear receptor TR4 in the etiology of Cushing disease

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

    doi: 10.1073/pnas.1306182110

    TR4-mediated POMC regulation is altered by ERK-mediated phosphorylation. ( A ) AtT20 cells were cotransfected with a POMC promoter-luciferase reporter plasmid and either the TR4 expression plasmid mTR4V5 or a control vector, after which they were treated
    Figure Legend Snippet: TR4-mediated POMC regulation is altered by ERK-mediated phosphorylation. ( A ) AtT20 cells were cotransfected with a POMC promoter-luciferase reporter plasmid and either the TR4 expression plasmid mTR4V5 or a control vector, after which they were treated

    Techniques Used: Luciferase, Plasmid Preparation, Expressing

    Athymic nude mice were inoculated subcutaneously with corticotroph tumor AtT20 cells (1 × 10 5 cells per mouse with 0.1 mL matrigel) either stably overexpressing TR4 plasmid mTR4V5H6 (TR4) or the pcDNAV5HisA control vector (Ctrl). ( A ) Tumor growth
    Figure Legend Snippet: Athymic nude mice were inoculated subcutaneously with corticotroph tumor AtT20 cells (1 × 10 5 cells per mouse with 0.1 mL matrigel) either stably overexpressing TR4 plasmid mTR4V5H6 (TR4) or the pcDNAV5HisA control vector (Ctrl). ( A ) Tumor growth

    Techniques Used: Mouse Assay, Stable Transfection, Plasmid Preparation

    TR4 regulates POMC expression and ACTH secretion in murine and human corticotroph tumor cells by directly binding to POMC. ( A ) AtT20 cells cotransfected with a rat POMC promoter-luciferase reporter plasmid (JA300) and control vector (pcDNAV5H6) or a TR4
    Figure Legend Snippet: TR4 regulates POMC expression and ACTH secretion in murine and human corticotroph tumor cells by directly binding to POMC. ( A ) AtT20 cells cotransfected with a rat POMC promoter-luciferase reporter plasmid (JA300) and control vector (pcDNAV5H6) or a TR4

    Techniques Used: Expressing, Binding Assay, Luciferase, Plasmid Preparation

    TR4 knockdown inhibits in vivo murine corticotroph tumor growth. Athymic nude mice were inoculated s.c. with either TR4 stable knockdown (shTR4-621) or nonsilencing shRNA-infected (NS shRNA) murine corticotroph tumor AtT20 cells (4 × 10 5 cells
    Figure Legend Snippet: TR4 knockdown inhibits in vivo murine corticotroph tumor growth. Athymic nude mice were inoculated s.c. with either TR4 stable knockdown (shTR4-621) or nonsilencing shRNA-infected (NS shRNA) murine corticotroph tumor AtT20 cells (4 × 10 5 cells

    Techniques Used: In Vivo, Mouse Assay, shRNA, Infection

    Transient or stable TR4 knockdown inhibits, whereas TR4 overexpression increases corticotroph tumor cell proliferation and invasion. Relative proliferation rates in murine corticotroph tumor AtT20 cells following either ( A ) transient transfection of a
    Figure Legend Snippet: Transient or stable TR4 knockdown inhibits, whereas TR4 overexpression increases corticotroph tumor cell proliferation and invasion. Relative proliferation rates in murine corticotroph tumor AtT20 cells following either ( A ) transient transfection of a

    Techniques Used: Over Expression, Transfection

    17) Product Images from "Selective estrogen receptor modulators decrease invasiveness in pituitary adenoma cell lines AtT‐20 and TtT/GF by affecting expression of MMP‐14 and ADAM12"

    Article Title: Selective estrogen receptor modulators decrease invasiveness in pituitary adenoma cell lines AtT‐20 and TtT/GF by affecting expression of MMP‐14 and ADAM12

    Journal: FEBS Open Bio

    doi: 10.1002/2211-5463.12999

    Selective estrogen receptor modulators reduce invasiveness in PA cell lines AtT‐20 (B) and TtT/GF (C). (A) Representative images of AtT20 (upper panel) and TtT/GF (lower panel) cells stained with DAPI. Note that cell lines were treated with 25% inhibitory concentrations (IC 25 ) of SERMs bazedoxifene, clomiphene, and raloxifene as indicated in Table 1 . Scale bar in (A), for all images, 50 μm. Batimastat, a broad‐range MMP inhibitor, served as positive control. With bazedoxifene in TtT/GF cells, the outlier (o) was excluded from the statistical analysis. Values are given as median, first quartile, and third quartile. Values are obtained from three independent experiments performed in triplicates. t served as statistical test. *Statistically significant difference as compared to control.
    Figure Legend Snippet: Selective estrogen receptor modulators reduce invasiveness in PA cell lines AtT‐20 (B) and TtT/GF (C). (A) Representative images of AtT20 (upper panel) and TtT/GF (lower panel) cells stained with DAPI. Note that cell lines were treated with 25% inhibitory concentrations (IC 25 ) of SERMs bazedoxifene, clomiphene, and raloxifene as indicated in Table 1 . Scale bar in (A), for all images, 50 μm. Batimastat, a broad‐range MMP inhibitor, served as positive control. With bazedoxifene in TtT/GF cells, the outlier (o) was excluded from the statistical analysis. Values are given as median, first quartile, and third quartile. Values are obtained from three independent experiments performed in triplicates. t served as statistical test. *Statistically significant difference as compared to control.

    Techniques Used: Staining, Positive Control

    Transfection of AtT‐20 (A, B) and TtT/GF (C, D) cells with siRNA silencing ADAM12 and MMP‐14 genes and their effect on invasion. Western blots for the respective proteins in AtT‐20 (A) and TtT/GF (C) cells; scramble (‘scr’) siRNA served as transfection and tubulin as loading controls. Invasion rates after selective silencing of ADAM12 and MMP‐14 genes as compared to scramble siRNA in AtT‐20 (B) and TtT/GF cells (D) were calculated. Values are obtained from three independent experiments in triplicates and are indicated as median, first quartile, and third quartile. t served as statistical test. *Statistically significant difference as compared to control (Scramble).
    Figure Legend Snippet: Transfection of AtT‐20 (A, B) and TtT/GF (C, D) cells with siRNA silencing ADAM12 and MMP‐14 genes and their effect on invasion. Western blots for the respective proteins in AtT‐20 (A) and TtT/GF (C) cells; scramble (‘scr’) siRNA served as transfection and tubulin as loading controls. Invasion rates after selective silencing of ADAM12 and MMP‐14 genes as compared to scramble siRNA in AtT‐20 (B) and TtT/GF cells (D) were calculated. Values are obtained from three independent experiments in triplicates and are indicated as median, first quartile, and third quartile. t served as statistical test. *Statistically significant difference as compared to control (Scramble).

    Techniques Used: Transfection, Western Blot

    Western blot analyses of whole‐cell lysates from AtT‐20 cells (A, C) and TtT/GF cells (B, D) treated with 50% inhibitory concentrations (IC 50 , as indicated in Table 1 ) of SERMs bazedoxifene (B, red), clomiphene (C, green), and raloxifene (R, yellow) for three days. Protein levels of ADAM12 (A, B) and MMP‐14 (C, D) are shown from one representative experiment. Note that ADAM12 can be seen as 68‐kDa band (black arrowhead), whereas MMP‐14 is shown as a 66‐kDa band (black arrowhead) compared to solvent control (‘DMSO’). Blank served as an additional control to judge the vehicle effect. Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH, 35 kDa, white arrowhead) was used as loading control. Bands were quantified by imagej analysis from three quantifications. Values are given as mean ± SEM using one‐way ANOVA. t served as statistical test, with ** P
    Figure Legend Snippet: Western blot analyses of whole‐cell lysates from AtT‐20 cells (A, C) and TtT/GF cells (B, D) treated with 50% inhibitory concentrations (IC 50 , as indicated in Table 1 ) of SERMs bazedoxifene (B, red), clomiphene (C, green), and raloxifene (R, yellow) for three days. Protein levels of ADAM12 (A, B) and MMP‐14 (C, D) are shown from one representative experiment. Note that ADAM12 can be seen as 68‐kDa band (black arrowhead), whereas MMP‐14 is shown as a 66‐kDa band (black arrowhead) compared to solvent control (‘DMSO’). Blank served as an additional control to judge the vehicle effect. Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH, 35 kDa, white arrowhead) was used as loading control. Bands were quantified by imagej analysis from three quantifications. Values are given as mean ± SEM using one‐way ANOVA. t served as statistical test, with ** P

    Techniques Used: Western Blot

    Quantitative real‐time‐polymerase chain reaction to determine mRNA levels of ADAM12 in AtT‐20 (A), of Basigin in AtT‐20 cells (B), of ADAM12 in TtT/GF cells (C), of MMP‐14 in TtT/GF cells (D), and of basigin in TtT/GF cells (E) in response to SERM treatment. For each SERM (raloxifene, bazedoxifene, and clomiphene), 50% inhibitory concentrations (IC 50 ) as indicated in Table 1 were used to treat cells for 72 h. Quantitative PCR was performed in three independent experiments in triplicates. Values are given as median, first quartile, and third quartile. t served as statistical test. *Statistically significant difference as compared to control.
    Figure Legend Snippet: Quantitative real‐time‐polymerase chain reaction to determine mRNA levels of ADAM12 in AtT‐20 (A), of Basigin in AtT‐20 cells (B), of ADAM12 in TtT/GF cells (C), of MMP‐14 in TtT/GF cells (D), and of basigin in TtT/GF cells (E) in response to SERM treatment. For each SERM (raloxifene, bazedoxifene, and clomiphene), 50% inhibitory concentrations (IC 50 ) as indicated in Table 1 were used to treat cells for 72 h. Quantitative PCR was performed in three independent experiments in triplicates. Values are given as median, first quartile, and third quartile. t served as statistical test. *Statistically significant difference as compared to control.

    Techniques Used: Real-time Polymerase Chain Reaction

    Mean cycle number values as determined by qPCR representing absolute gene expression levels of MMP‐1, MMP‐9, MMP‐14, ADAM8, and ADAM12 in untreated PA cell lines AtT‐20 and TtT/GF.
    Figure Legend Snippet: Mean cycle number values as determined by qPCR representing absolute gene expression levels of MMP‐1, MMP‐9, MMP‐14, ADAM8, and ADAM12 in untreated PA cell lines AtT‐20 and TtT/GF.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    Dose–response curves for SERMs bazedoxifene, clomiphene, and raloxifene in PA cell lines AtT‐20 (A) and TtT/GF (B). Values are indicated as mean ± SEM. ctrl, control.
    Figure Legend Snippet: Dose–response curves for SERMs bazedoxifene, clomiphene, and raloxifene in PA cell lines AtT‐20 (A) and TtT/GF (B). Values are indicated as mean ± SEM. ctrl, control.

    Techniques Used:

    18) Product Images from "Inhibitory Effects of a Novel PPAR-γ Agonist MEKT1 on Pomc Expression/ACTH Secretion in AtT20 Cells"

    Article Title: Inhibitory Effects of a Novel PPAR-γ Agonist MEKT1 on Pomc Expression/ACTH Secretion in AtT20 Cells

    Journal: PPAR Research

    doi: 10.1155/2018/5346272

    MEKT1-mediated effect on AtT20 cell proliferation and apoptosis. (a) AtT20 cells were incubated for 96 hours either in the presence of MEKT1 (1 nM, 10 nM, 100 nM, 1 μ M, or 10 μ M) or DMSO (0.1%) as a control for 24 hours before assay. (b) AtT20 cells were incubated for 96 hours either in the presence of pioglitazone (1 nM, 10 nM, 100 nM, 1 μ M, or 10 μ M) or DMSO (0.1%) as a control for 24 hours before assay. (c) AtT20 cells were incubated for 96 hours either in the presence of rosiglitazone (1 nM, 10 nM, 100 nM, 1 μ M, or 10 μ M) or DMSO (0.1%) as a control for 24 hours before assay. Data are expressed as percentages (100%) of control. (d) Effects of MEKT1 on mRNA expression of m Pttg dose-dependently. AtT20 cells were treated with MEKT1 (1 nM, 10 nM, 100 nM, 1 μ M, or 10 μ M) or 0.1% DMSO (vehicle control) for 24 hours. (e) Effects on MEKT1 (10 μ M) on AtT20 cell apoptosis. Each point indicates mean ± SEM ( n = 4). NS stands for “not significant.”
    Figure Legend Snippet: MEKT1-mediated effect on AtT20 cell proliferation and apoptosis. (a) AtT20 cells were incubated for 96 hours either in the presence of MEKT1 (1 nM, 10 nM, 100 nM, 1 μ M, or 10 μ M) or DMSO (0.1%) as a control for 24 hours before assay. (b) AtT20 cells were incubated for 96 hours either in the presence of pioglitazone (1 nM, 10 nM, 100 nM, 1 μ M, or 10 μ M) or DMSO (0.1%) as a control for 24 hours before assay. (c) AtT20 cells were incubated for 96 hours either in the presence of rosiglitazone (1 nM, 10 nM, 100 nM, 1 μ M, or 10 μ M) or DMSO (0.1%) as a control for 24 hours before assay. Data are expressed as percentages (100%) of control. (d) Effects of MEKT1 on mRNA expression of m Pttg dose-dependently. AtT20 cells were treated with MEKT1 (1 nM, 10 nM, 100 nM, 1 μ M, or 10 μ M) or 0.1% DMSO (vehicle control) for 24 hours. (e) Effects on MEKT1 (10 μ M) on AtT20 cell apoptosis. Each point indicates mean ± SEM ( n = 4). NS stands for “not significant.”

    Techniques Used: Incubation, Expressing

    Involvement of PPAR- γ in the MEKT1 effects on Pomc mRNA expression. Effects of PPAR- γ knockdown by its siRNA on (a) PPAR-γ mRNA expression, (b) PPAR-α mRNA expression, and (c) PPAR-β mRNA expression. AtT20 cells transiently transfected with siRNA (negative control; NC or PPAR- γ ) for 48 hours were incubated with 0.1% DMSO (control) for 24 hours. Results are expressed as percentages of each control. Each point represents mean ± SEM ( n = 4). ∗ P
    Figure Legend Snippet: Involvement of PPAR- γ in the MEKT1 effects on Pomc mRNA expression. Effects of PPAR- γ knockdown by its siRNA on (a) PPAR-γ mRNA expression, (b) PPAR-α mRNA expression, and (c) PPAR-β mRNA expression. AtT20 cells transiently transfected with siRNA (negative control; NC or PPAR- γ ) for 48 hours were incubated with 0.1% DMSO (control) for 24 hours. Results are expressed as percentages of each control. Each point represents mean ± SEM ( n = 4). ∗ P

    Techniques Used: Expressing, Transfection, Negative Control, Incubation

    Effects of MEKT1, rosiglitazone, and pioglitazone on Pomc promoter activity in AtT20 cells. AtT20 cells transiently transfected with 300 ng full length r Pomc -Luc (−703/+58-luc) and 100 ng pRSV- β -gal were treated with MEKT1, rosiglitazone (Rosi), and pioglitazone (Pio) (1 nM, 10 nM, 100 nM, 1 μ M, or 10 μ M) or 0.1% DMSO (vehicle control) for 24 hours. Data are expressed as percentages (100%) of control. Each point represents mean ± SEM ( n = 4). ∗∗ P
    Figure Legend Snippet: Effects of MEKT1, rosiglitazone, and pioglitazone on Pomc promoter activity in AtT20 cells. AtT20 cells transiently transfected with 300 ng full length r Pomc -Luc (−703/+58-luc) and 100 ng pRSV- β -gal were treated with MEKT1, rosiglitazone (Rosi), and pioglitazone (Pio) (1 nM, 10 nM, 100 nM, 1 μ M, or 10 μ M) or 0.1% DMSO (vehicle control) for 24 hours. Data are expressed as percentages (100%) of control. Each point represents mean ± SEM ( n = 4). ∗∗ P

    Techniques Used: Activity Assay, Transfection

    Effects of MEKT1 on the interaction between Nur77/Nurr1 and NurRE, Tpit and TpitRE, and Nur77 and NBRE on Pomc promoter (a) in AtT20 cells. Effects of MEKT1 on the interaction between Nur77/Nurr1 and NurRE (b), Tpit and TpitRE (c), and Nur77 and NBRE (d) on Pomc promoter examined by ChIP assay using NurRE, TpitRE, and NBRE primer. ChIP assay was carried out using digested chromatin extracted from the cells cultured in the presence of either 10 μ M MEKT1 or 0.1% DMSO (control) for 24 hours. Chromatin fragments were immunoprecipitated either by normal rabbit IgG (negative control), anti-Nur77/Nurr1 antibody, or anti-Tpit (anti TBX 19) antibody. Purified DNA was analyzed by qPCR using primers specific for NurRE, TpitRE, and NBRE containing sequence on Pomc promoter. The primer product sizes of NurRE, TpitRE, and NBRE were 211 bp, 146 bp, and 102 bp, respectively. Immunoprecipitated DNA was amplified by qPCR and then normalized to the values obtained after amplification of immunoprecipitated 1% input DNA. Data represent mean ± SEM ( n = 3). NS means “not significant.” ∗ P
    Figure Legend Snippet: Effects of MEKT1 on the interaction between Nur77/Nurr1 and NurRE, Tpit and TpitRE, and Nur77 and NBRE on Pomc promoter (a) in AtT20 cells. Effects of MEKT1 on the interaction between Nur77/Nurr1 and NurRE (b), Tpit and TpitRE (c), and Nur77 and NBRE (d) on Pomc promoter examined by ChIP assay using NurRE, TpitRE, and NBRE primer. ChIP assay was carried out using digested chromatin extracted from the cells cultured in the presence of either 10 μ M MEKT1 or 0.1% DMSO (control) for 24 hours. Chromatin fragments were immunoprecipitated either by normal rabbit IgG (negative control), anti-Nur77/Nurr1 antibody, or anti-Tpit (anti TBX 19) antibody. Purified DNA was analyzed by qPCR using primers specific for NurRE, TpitRE, and NBRE containing sequence on Pomc promoter. The primer product sizes of NurRE, TpitRE, and NBRE were 211 bp, 146 bp, and 102 bp, respectively. Immunoprecipitated DNA was amplified by qPCR and then normalized to the values obtained after amplification of immunoprecipitated 1% input DNA. Data represent mean ± SEM ( n = 3). NS means “not significant.” ∗ P

    Techniques Used: Chromatin Immunoprecipitation, Cell Culture, Immunoprecipitation, Negative Control, Purification, Real-time Polymerase Chain Reaction, Sequencing, Amplification

    Effects of Nur77, Tpit, and Nurr1 overexpression on MEKT1-mediated effect of Pomc mRNA expression. (a) Nur77 overexpression effect on the MEKT1-mediated suppression of Pomc mRNA expression in AtT20 cells. AtT20 cells transiently transfected with pcDNA3 and Nur77 overexpression plasmid were incubated either in the presence of MEKT1 at 10 μ M or DMSO at 0.1% (control) for 24 hours. (b) Tpit overexpression effect on the MEKT1-mediated suppression of Pomc mRNA expression. AtT20 cells transiently transfected with pcDNA3 and Tpit overexpression plasmid were incubated either in the presence of MEKT1 at 10 μ M or DMSO at 0.1% (control) for 24 hours. (c) Nurr1 overexpression on the MEKT1-mediated suppression of Pomc mRNA expression. AtT20 cells transiently transfected with pcDNA3 and Nurr1 overexpression plasmid were incubated either in the presence of MEKT1 at 10 μ M or DMSO at 0.1% (control) for 24 hours. Each overexpression plasmid volume was maintained to 300 ng adding pcDNA3 empty vector. Results are expressed as percentages (100%) of control. Data represent mean ± SEM ( n = 4). NS stands for “not significant.” ∗ P
    Figure Legend Snippet: Effects of Nur77, Tpit, and Nurr1 overexpression on MEKT1-mediated effect of Pomc mRNA expression. (a) Nur77 overexpression effect on the MEKT1-mediated suppression of Pomc mRNA expression in AtT20 cells. AtT20 cells transiently transfected with pcDNA3 and Nur77 overexpression plasmid were incubated either in the presence of MEKT1 at 10 μ M or DMSO at 0.1% (control) for 24 hours. (b) Tpit overexpression effect on the MEKT1-mediated suppression of Pomc mRNA expression. AtT20 cells transiently transfected with pcDNA3 and Tpit overexpression plasmid were incubated either in the presence of MEKT1 at 10 μ M or DMSO at 0.1% (control) for 24 hours. (c) Nurr1 overexpression on the MEKT1-mediated suppression of Pomc mRNA expression. AtT20 cells transiently transfected with pcDNA3 and Nurr1 overexpression plasmid were incubated either in the presence of MEKT1 at 10 μ M or DMSO at 0.1% (control) for 24 hours. Each overexpression plasmid volume was maintained to 300 ng adding pcDNA3 empty vector. Results are expressed as percentages (100%) of control. Data represent mean ± SEM ( n = 4). NS stands for “not significant.” ∗ P

    Techniques Used: Over Expression, Expressing, Transfection, Plasmid Preparation, Incubation

    MEKT1-mediated effects on the mRNA expression of mouse Nur77 , Nurr1 , NeuroD1 , Tpit , Pitx , NFkB1 , and NFkB2 in AtT20 cells. AtT20 cells treated with MEKT1 (10 μ M) or 0.1% DMSO (vehicle control) for 24 hours. (a) Nur77 mRNA expression, (b) Nurr1 mRNA expression, (c) NeuroD1 mRNA expression, (d) Tpit mRNA expression, (e) Pitx mRNA expression, (f) NFκB1 mRNA expression, and (g) NFκB2 mRNA expression. Data are expressed as percentages (100%) of control. Data represent mean ± SEM ( n = 4). ∗∗∗ P
    Figure Legend Snippet: MEKT1-mediated effects on the mRNA expression of mouse Nur77 , Nurr1 , NeuroD1 , Tpit , Pitx , NFkB1 , and NFkB2 in AtT20 cells. AtT20 cells treated with MEKT1 (10 μ M) or 0.1% DMSO (vehicle control) for 24 hours. (a) Nur77 mRNA expression, (b) Nurr1 mRNA expression, (c) NeuroD1 mRNA expression, (d) Tpit mRNA expression, (e) Pitx mRNA expression, (f) NFκB1 mRNA expression, and (g) NFκB2 mRNA expression. Data are expressed as percentages (100%) of control. Data represent mean ± SEM ( n = 4). ∗∗∗ P

    Techniques Used: Expressing

    Effects of MEKT1 (time dependently), rosiglitazone, and pioglitazone on Nurr1, Nur77, and Tpit protein expression. (a) AtT20 cells treated with MEKT1 (M) at 10 μ M for 24 hours, 6 hours, and 3 hours, rosiglitazone (R) at 10 μ M for 24 hours, and pioglitazone (P) at 10 μ M for 24 hours, or 0.1% DMSO as control (C) for 24 hours. Optical density (OD) of Nurr1/Nur77 was shown in figure (b), Nur77 in figure (c), and TBX19 (Tpit) in figure (d). OD of Nurr1/Nur77, Nur77, and TBX19 (Tpit) were normalized by OD of actin. Results are expressed as percentages of control (100%).
    Figure Legend Snippet: Effects of MEKT1 (time dependently), rosiglitazone, and pioglitazone on Nurr1, Nur77, and Tpit protein expression. (a) AtT20 cells treated with MEKT1 (M) at 10 μ M for 24 hours, 6 hours, and 3 hours, rosiglitazone (R) at 10 μ M for 24 hours, and pioglitazone (P) at 10 μ M for 24 hours, or 0.1% DMSO as control (C) for 24 hours. Optical density (OD) of Nurr1/Nur77 was shown in figure (b), Nur77 in figure (c), and TBX19 (Tpit) in figure (d). OD of Nurr1/Nur77, Nur77, and TBX19 (Tpit) were normalized by OD of actin. Results are expressed as percentages of control (100%).

    Techniques Used: Expressing

    Effects of MEKT1, rosiglitazone, and pioglitazone on ACTH secretion from AtT20 cells. (a) Effects of MEKT1, rosiglitazone, and pioglitazone on ACTH secretion into the media from AtT20 cells. AtT20 cells were treated with MEKT1, rosiglitazone (Rosi), and pioglitazone (Pio) (10 μ M) or DMSO (0.1%) as a control. After 24-hour incubation of the cells, the ACTH secreted to the media was determined by EIA. (b) Dose-dependent effects of MEKT1 on ACTH secretion into the media from AtT20 cells. AtT20 cells were treated with MEKT1 (10 nM, 100 nM, 1 μ M, or 10 μ M) or 0.1% DMSO (vehicle control) for 24 hours. After 24-hour incubation of the cells, the ACTH secreted to the media was determined by EIA. Data are expressed as percentages (100%) of control. Each point represents mean ± SEM ( n = 4). NS means “not significant.” ∗∗∗ P
    Figure Legend Snippet: Effects of MEKT1, rosiglitazone, and pioglitazone on ACTH secretion from AtT20 cells. (a) Effects of MEKT1, rosiglitazone, and pioglitazone on ACTH secretion into the media from AtT20 cells. AtT20 cells were treated with MEKT1, rosiglitazone (Rosi), and pioglitazone (Pio) (10 μ M) or DMSO (0.1%) as a control. After 24-hour incubation of the cells, the ACTH secreted to the media was determined by EIA. (b) Dose-dependent effects of MEKT1 on ACTH secretion into the media from AtT20 cells. AtT20 cells were treated with MEKT1 (10 nM, 100 nM, 1 μ M, or 10 μ M) or 0.1% DMSO (vehicle control) for 24 hours. After 24-hour incubation of the cells, the ACTH secreted to the media was determined by EIA. Data are expressed as percentages (100%) of control. Each point represents mean ± SEM ( n = 4). NS means “not significant.” ∗∗∗ P

    Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay

    Effects of MEKT1, rosiglitazone, and pioglitazone on mRNA expression of Pomc in AtT20 cells. (a) Effects of MEKT1, rosiglitazone, and pioglitazone on mRNA expression of Pomc dose-dependently. AtT20 cells were treated with MEKT1, rosiglitazone (Rosi), and pioglitazone (Pio) (1 nM, 10 nM, 100 nM, 1 μ M, or 10 μ M) or 0.1% DMSO (vehicle control) for 24 hours. ∗ P
    Figure Legend Snippet: Effects of MEKT1, rosiglitazone, and pioglitazone on mRNA expression of Pomc in AtT20 cells. (a) Effects of MEKT1, rosiglitazone, and pioglitazone on mRNA expression of Pomc dose-dependently. AtT20 cells were treated with MEKT1, rosiglitazone (Rosi), and pioglitazone (Pio) (1 nM, 10 nM, 100 nM, 1 μ M, or 10 μ M) or 0.1% DMSO (vehicle control) for 24 hours. ∗ P

    Techniques Used: Expressing

    Effects of MEKT1 on Pomc promoter deletion mutants and role of NurRE, TpitRE, and NBRE on MEKT1-mediated effect on Pomc promoter activity in AtT20 cells. (a) MEKT1-mediated effect on Pomc promoter deletion mutants. AtT20 cells transiently transfected with 300 ng rPomc-Luc (−703/+58-luc) or each deletion mutant reporter plasmid (−429/+58-Luc, −379/+58-Luc, −359/+58-Luc, −293/+58, −169/+58, and +12/+58) and 100 ng pRSV- β -gal were incubated in the presence (10 μ M) or absence of MEKT1 for 24 hours before the luciferase assay. Data are expressed as percentages of each control (100% in pGL3-Basic). (b) MEKT1-mediated effect on Pomc promoter activity using NurRE mut, TpitRE mut, and NBRE mut. AtT20 cells transiently transfected with 300 ng r Pomc -Luc (−703/+58-luc) or NurRE mut (r Pomc -Luc- NurRE -Mut), TpitRE mutant (r Pomc -Luc- TpitRE -Mut), NBRE mutant of Pomc promoter (r Pomc -Luc- NBRE -Mut) of Pomc full length promoter and 150 ng pRSV- β -gal were incubated in the presence (10 μ M) or absence of MEKT1 for 24 hours before the luciferase assay. Data are expressed as percentages of each control (100% in r Pomc -Luc). Data represent mean ± SEM ( n = 4). NS denotes “not significant.” ∗ P
    Figure Legend Snippet: Effects of MEKT1 on Pomc promoter deletion mutants and role of NurRE, TpitRE, and NBRE on MEKT1-mediated effect on Pomc promoter activity in AtT20 cells. (a) MEKT1-mediated effect on Pomc promoter deletion mutants. AtT20 cells transiently transfected with 300 ng rPomc-Luc (−703/+58-luc) or each deletion mutant reporter plasmid (−429/+58-Luc, −379/+58-Luc, −359/+58-Luc, −293/+58, −169/+58, and +12/+58) and 100 ng pRSV- β -gal were incubated in the presence (10 μ M) or absence of MEKT1 for 24 hours before the luciferase assay. Data are expressed as percentages of each control (100% in pGL3-Basic). (b) MEKT1-mediated effect on Pomc promoter activity using NurRE mut, TpitRE mut, and NBRE mut. AtT20 cells transiently transfected with 300 ng r Pomc -Luc (−703/+58-luc) or NurRE mut (r Pomc -Luc- NurRE -Mut), TpitRE mutant (r Pomc -Luc- TpitRE -Mut), NBRE mutant of Pomc promoter (r Pomc -Luc- NBRE -Mut) of Pomc full length promoter and 150 ng pRSV- β -gal were incubated in the presence (10 μ M) or absence of MEKT1 for 24 hours before the luciferase assay. Data are expressed as percentages of each control (100% in r Pomc -Luc). Data represent mean ± SEM ( n = 4). NS denotes “not significant.” ∗ P

    Techniques Used: Activity Assay, Transfection, Mutagenesis, Plasmid Preparation, Incubation, Luciferase

    19) Product Images from "Driver mutations in USP8 wild-type Cushing’s disease"

    Article Title: Driver mutations in USP8 wild-type Cushing’s disease

    Journal: Neuro-Oncology

    doi: 10.1093/neuonc/noz109

    USP48 potentiates CRH-induced POMC promoter activity and ACTH secretion. (A) Cell viability in AtT-20 cells transfected with USP48 wt and Met415Ile mutant. (B) Basal POMC promoter activity presented as percentage of empty vector (mock) control. Data are luciferase/β-galactosidase ratio, means of 3 experiments with each transfection condition done in triplicates. (C) Basal ACTH secretion in cells overexpressing USP48 wt and mutant determined by radioimmunoassay and expressed as percentage of empty vector control. Means of 7 experiments, with each condition in each experiment done in quadruplicates. * P
    Figure Legend Snippet: USP48 potentiates CRH-induced POMC promoter activity and ACTH secretion. (A) Cell viability in AtT-20 cells transfected with USP48 wt and Met415Ile mutant. (B) Basal POMC promoter activity presented as percentage of empty vector (mock) control. Data are luciferase/β-galactosidase ratio, means of 3 experiments with each transfection condition done in triplicates. (C) Basal ACTH secretion in cells overexpressing USP48 wt and mutant determined by radioimmunoassay and expressed as percentage of empty vector control. Means of 7 experiments, with each condition in each experiment done in quadruplicates. * P

    Techniques Used: Activity Assay, Transfection, Mutagenesis, Plasmid Preparation, Luciferase, RIA Assay

    20) Product Images from "Cannabichromene is a cannabinoid CB2 receptor agonist. Cannabichromene is a cannabinoid CB2 receptor agonist"

    Article Title: Cannabichromene is a cannabinoid CB2 receptor agonist. Cannabichromene is a cannabinoid CB2 receptor agonist

    Journal: British Journal of Pharmacology

    doi: 10.1111/bph.14815

    Desensitization of AtT20‐CB2 cells signalling. (a) A representative trace of 1‐μM CP55,940 desensitization of AtT20‐CB2 cell signalling in the presence of vehicle or Compound 101. (b) A representative trace of 10‐μM CBC desensitization of AtT20‐CB2 cell signalling in the presence of vehicle or Compound 101. Cells were pre‐incubated with Compound 101 (10‐μM, 60 min) before CP55,940 or CBC addition. CP55,940 or CBC were added after 2 min of baseline reading and read for 30 min. (c) Summary data of CBC (10‐μM, 30 min) desensitization of AtT20‐CB 2 receptor signalling. Peak hyperpolarization was determined within 5 min of drug addition, and peak depolarization was determined at 30 min of drug addition. All data are expressed as mean change in fluorescence due to cellular hyperpolarization ± SEM, after subtraction of baseline ( n = 6). * P
    Figure Legend Snippet: Desensitization of AtT20‐CB2 cells signalling. (a) A representative trace of 1‐μM CP55,940 desensitization of AtT20‐CB2 cell signalling in the presence of vehicle or Compound 101. (b) A representative trace of 10‐μM CBC desensitization of AtT20‐CB2 cell signalling in the presence of vehicle or Compound 101. Cells were pre‐incubated with Compound 101 (10‐μM, 60 min) before CP55,940 or CBC addition. CP55,940 or CBC were added after 2 min of baseline reading and read for 30 min. (c) Summary data of CBC (10‐μM, 30 min) desensitization of AtT20‐CB 2 receptor signalling. Peak hyperpolarization was determined within 5 min of drug addition, and peak depolarization was determined at 30 min of drug addition. All data are expressed as mean change in fluorescence due to cellular hyperpolarization ± SEM, after subtraction of baseline ( n = 6). * P

    Techniques Used: Incubation, Fluorescence

    CBC activation of CB 2 receptors is blocked by AM630. (a) A representative trace of change in fluorescence of AtT20‐CB2 cells after 5‐min pretreatment with vehicle and 3‐μM AM630, followed by the addition of 10‐μM CBC. (b) Responses to CBC (10‐μM) and CP55,940 (300 nM) in AtT20‐CB2 cells with or without pre‐incubation of AM630 (3‐μM) for 5 min. Results are expressed as mean ± SEM ( n = 5). * P
    Figure Legend Snippet: CBC activation of CB 2 receptors is blocked by AM630. (a) A representative trace of change in fluorescence of AtT20‐CB2 cells after 5‐min pretreatment with vehicle and 3‐μM AM630, followed by the addition of 10‐μM CBC. (b) Responses to CBC (10‐μM) and CP55,940 (300 nM) in AtT20‐CB2 cells with or without pre‐incubation of AM630 (3‐μM) for 5 min. Results are expressed as mean ± SEM ( n = 5). * P

    Techniques Used: Activation Assay, Fluorescence, Incubation

    CBC activates CB 2 , but not CB 1 , receptors (a) Concentration‐response curves of CP55,940, THC, and CBC in AtT20‐CB1 cells ( n = 5). Results are expressed as mean ± SEM after normalization to 1‐μM CP55,940 hyperpolarization. (b) Representative traces of changes in fluorescence, due to CBC and 1‐μM CP55,940‐induced hyperpolarization in AtT20‐CB1 cells. Drugs were added after 120 s of baseline reading and read over 300 s. (c) Concentration‐response curves of CP55,940, THC, and CBC in AtT20‐CB2 cells ( n = 5). Results are expressed as mean ± SEM after normalization to 1‐μM CP55,940 hyperpolarization. (d) Representative traces of changes in fluorescence, due to CBC and 1‐μM CP55,940‐induced hyperpolarization in AtT20‐CB2 cells. Drugs were added after 120 s of baseline reading and read over 300 s
    Figure Legend Snippet: CBC activates CB 2 , but not CB 1 , receptors (a) Concentration‐response curves of CP55,940, THC, and CBC in AtT20‐CB1 cells ( n = 5). Results are expressed as mean ± SEM after normalization to 1‐μM CP55,940 hyperpolarization. (b) Representative traces of changes in fluorescence, due to CBC and 1‐μM CP55,940‐induced hyperpolarization in AtT20‐CB1 cells. Drugs were added after 120 s of baseline reading and read over 300 s. (c) Concentration‐response curves of CP55,940, THC, and CBC in AtT20‐CB2 cells ( n = 5). Results are expressed as mean ± SEM after normalization to 1‐μM CP55,940 hyperpolarization. (d) Representative traces of changes in fluorescence, due to CBC and 1‐μM CP55,940‐induced hyperpolarization in AtT20‐CB2 cells. Drugs were added after 120 s of baseline reading and read over 300 s

    Techniques Used: Concentration Assay, Fluorescence

    CBC antagonizes CP55,940 and THC response in CB2 cells. Representative traces of the effect of CBC (10‐μM) on (a) CP55,940 (100 nM) on fluorescence in AtT20‐CB1 cells loaded with a membrane potential‐sensitive dye. (b) THC (10‐μM) hyperpolarization in AtT20‐CB1. (c) CP55,940 (300 nM) hyperpolarization in AtT20‐CB2 cells. After 2‐min baseline reading, cells were pretreated with vehicle or 10‐μM CBC for 5 min, followed by the addition CP55,940. (d) Summary data of the effect of 10‐μM CBC on 300‐nM CP55,940 in AtT20‐CB2 cells. Results are expressed as mean ± SEM after normalization to 1‐μM CP55,940 hyperpolarization ( n = 5. * P
    Figure Legend Snippet: CBC antagonizes CP55,940 and THC response in CB2 cells. Representative traces of the effect of CBC (10‐μM) on (a) CP55,940 (100 nM) on fluorescence in AtT20‐CB1 cells loaded with a membrane potential‐sensitive dye. (b) THC (10‐μM) hyperpolarization in AtT20‐CB1. (c) CP55,940 (300 nM) hyperpolarization in AtT20‐CB2 cells. After 2‐min baseline reading, cells were pretreated with vehicle or 10‐μM CBC for 5 min, followed by the addition CP55,940. (d) Summary data of the effect of 10‐μM CBC on 300‐nM CP55,940 in AtT20‐CB2 cells. Results are expressed as mean ± SEM after normalization to 1‐μM CP55,940 hyperpolarization ( n = 5. * P

    Techniques Used: Fluorescence

    21) Product Images from "Deducing corticotropin-releasing hormone receptor type 1 signaling networks from gene expression data by usage of genetic algorithms and graphical Gaussian models"

    Article Title: Deducing corticotropin-releasing hormone receptor type 1 signaling networks from gene expression data by usage of genetic algorithms and graphical Gaussian models

    Journal: BMC Systems Biology

    doi: 10.1186/1752-0509-4-159

    qRT-PCR validation of the differential expression of six candidate genes at different time points . Filled circles located on solid lines represent differential expression values from the microarray whereas filled squares on dashed lines show qRT-PCR expression values of AtT-20 cells independently treated with CRH related to their untreated controls and normalized to the house keeping gene Hprt. p-values were evaluated by ANOVA analyses.
    Figure Legend Snippet: qRT-PCR validation of the differential expression of six candidate genes at different time points . Filled circles located on solid lines represent differential expression values from the microarray whereas filled squares on dashed lines show qRT-PCR expression values of AtT-20 cells independently treated with CRH related to their untreated controls and normalized to the house keeping gene Hprt. p-values were evaluated by ANOVA analyses.

    Techniques Used: Quantitative RT-PCR, Expressing, Microarray

    22) Product Images from "TRPA1 regulates gastrointestinal motility through serotonin release from enterochromaffin cells"

    Article Title: TRPA1 regulates gastrointestinal motility through serotonin release from enterochromaffin cells

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

    doi: 10.1073/pnas.0805323106

    Analyses for TRPA1 agonist-induced 5-HT release mechanisms using TRPA1-specific siRNA. Effects of siRNA on either TRPA1 mRNA expression ( A ) or TRPA1 agonist-induced [Ca 2+ ] i ( B ) in RIN14B cells. ( C ) Effects of siRNA on TRPA1 agonist-induced 5-HT released from RIN14B cells. Three to 5 independent experiments were performed and typical results are shown, respectively. Data represent the mean ( B ) or the mean ± S.D. ( n = 3) ( A and C ), respectively. ***, P
    Figure Legend Snippet: Analyses for TRPA1 agonist-induced 5-HT release mechanisms using TRPA1-specific siRNA. Effects of siRNA on either TRPA1 mRNA expression ( A ) or TRPA1 agonist-induced [Ca 2+ ] i ( B ) in RIN14B cells. ( C ) Effects of siRNA on TRPA1 agonist-induced 5-HT released from RIN14B cells. Three to 5 independent experiments were performed and typical results are shown, respectively. Data represent the mean ( B ) or the mean ± S.D. ( n = 3) ( A and C ), respectively. ***, P

    Techniques Used: Expressing

    Stimulated 5-HT release and intracellular Ca 2+ in RIN14B cells. Ionomycin (10 μM) induced the release of 5-HT from RIN14B cells ( A ). The dose-response curves of TRPA1 agonist-induced increase in [Ca 2+ ] i in RIN14B cells ( B ). Blockade of the AITC (30 μM)-evoked increase in [Ca 2+ ] i induced by ruthenium red (3 μM) in RIN14B cells ( C ). TRPA1 agonist-induced release of 5-HT from RIN14B cells ( D ). Effects of the TRPA1 inhibitor ruthenium red (30 μM) on the AITC (300 μM)- or CA (300 μM)-induced 5-HT release from RIN14B cells ( E ). Role of Ca 2+ in the TRPA1 agonist-induced release of 5-HT from RIN14B cells ( F ). The cells were stimulated by AITC (300 μM) or CA (300 μM) in HBSS (open column) and Ca 2+ -free HBSS (solid column). All data represent the mean ± S.D. ( n = 3). **, P
    Figure Legend Snippet: Stimulated 5-HT release and intracellular Ca 2+ in RIN14B cells. Ionomycin (10 μM) induced the release of 5-HT from RIN14B cells ( A ). The dose-response curves of TRPA1 agonist-induced increase in [Ca 2+ ] i in RIN14B cells ( B ). Blockade of the AITC (30 μM)-evoked increase in [Ca 2+ ] i induced by ruthenium red (3 μM) in RIN14B cells ( C ). TRPA1 agonist-induced release of 5-HT from RIN14B cells ( D ). Effects of the TRPA1 inhibitor ruthenium red (30 μM) on the AITC (300 μM)- or CA (300 μM)-induced 5-HT release from RIN14B cells ( E ). Role of Ca 2+ in the TRPA1 agonist-induced release of 5-HT from RIN14B cells ( F ). The cells were stimulated by AITC (300 μM) or CA (300 μM) in HBSS (open column) and Ca 2+ -free HBSS (solid column). All data represent the mean ± S.D. ( n = 3). **, P

    Techniques Used:

    23) Product Images from "Identification of driver genes and key pathways of non-functional pituitary adenomas predicts the therapeutic effect of STO-609"

    Article Title: Identification of driver genes and key pathways of non-functional pituitary adenomas predicts the therapeutic effect of STO-609

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0240230

    (A) Cellular viability of HP75, GT1-1 and AtT-20 cells treated with STO-609 (B) Clonogenicities in Petri dishes with different doses of STO-609 (C) Scratch assay in control and STO-609 group (D) numbers of clone formation in HP75 cell line and GT1-1 cell line (E) Wound width in control and STO-609 (F) The distribution of cells in apoptosis with different doses of STO-609 (G) The percentage of apoptosis cell treated with different dose of STO-609.
    Figure Legend Snippet: (A) Cellular viability of HP75, GT1-1 and AtT-20 cells treated with STO-609 (B) Clonogenicities in Petri dishes with different doses of STO-609 (C) Scratch assay in control and STO-609 group (D) numbers of clone formation in HP75 cell line and GT1-1 cell line (E) Wound width in control and STO-609 (F) The distribution of cells in apoptosis with different doses of STO-609 (G) The percentage of apoptosis cell treated with different dose of STO-609.

    Techniques Used: Wound Healing Assay

    24) Product Images from "Role of NeuroD1 on the negative regulation of Pomc expression by glucocorticoid"

    Article Title: Role of NeuroD1 on the negative regulation of Pomc expression by glucocorticoid

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0175435

    Effects of DEX on NeuroD1 protein expression in AtT20 cells. (A) Time-dependent effect of DEX on NeuroD1 and actin protein expression. AtT20 cells were treated with DEX (100 nM) for 1 hr, 3 hrs, 6 hrs, 9 hrs, or 24 hrs before the Western blot analyses. Vehicle control: 0.1% ethanol. In (B), AtT20 cells were treated with DEX (100 nM) for 9 hrs or 24 hrs before the Western blot analyses. Vehicle control: 0.1% ethanol. Optical density (OD) of NeuroD1 was normalized by OD of actin. Results are expressed as percentages of control (100%) Each point represents mean ± SEM (n = 3). ** P
    Figure Legend Snippet: Effects of DEX on NeuroD1 protein expression in AtT20 cells. (A) Time-dependent effect of DEX on NeuroD1 and actin protein expression. AtT20 cells were treated with DEX (100 nM) for 1 hr, 3 hrs, 6 hrs, 9 hrs, or 24 hrs before the Western blot analyses. Vehicle control: 0.1% ethanol. In (B), AtT20 cells were treated with DEX (100 nM) for 9 hrs or 24 hrs before the Western blot analyses. Vehicle control: 0.1% ethanol. Optical density (OD) of NeuroD1 was normalized by OD of actin. Results are expressed as percentages of control (100%) Each point represents mean ± SEM (n = 3). ** P

    Techniques Used: Expressing, Western Blot

    Effects of DEX on interaction between NeuroD1 and E-box on Pomc promoter in AtT20 cells. (A) Effect of of DEX on interaction between NeuroD1 and E-box on Pomc promoter examined by ChIP assay using E-box primer. ChIP assay was performed using digested chromatin extracted from the cells cultured in the presence (100 nM) or absence (control) of DEX for 30 min (B), 60 min (C), or 24 hrs (D). Chromatin fragments were immunoprecipitated either by normal rabbit IgG (negative control) or NeuroD1 antibody. Purified DNA was analyzed by qPCR using primers specific for E-box containing sequence on Pomc promoter. The expected size of E-box is 196 bp. Few qPCR products observed in the input samples were detected in the immunoprecipitation using normal IgG. Immunoprecipitated DNA was quantified by qPCR and normalized to the values obtained after amplification of unprecipitated 1% input DNA. Each point represents mean ± SEM (n = 3). **P
    Figure Legend Snippet: Effects of DEX on interaction between NeuroD1 and E-box on Pomc promoter in AtT20 cells. (A) Effect of of DEX on interaction between NeuroD1 and E-box on Pomc promoter examined by ChIP assay using E-box primer. ChIP assay was performed using digested chromatin extracted from the cells cultured in the presence (100 nM) or absence (control) of DEX for 30 min (B), 60 min (C), or 24 hrs (D). Chromatin fragments were immunoprecipitated either by normal rabbit IgG (negative control) or NeuroD1 antibody. Purified DNA was analyzed by qPCR using primers specific for E-box containing sequence on Pomc promoter. The expected size of E-box is 196 bp. Few qPCR products observed in the input samples were detected in the immunoprecipitation using normal IgG. Immunoprecipitated DNA was quantified by qPCR and normalized to the values obtained after amplification of unprecipitated 1% input DNA. Each point represents mean ± SEM (n = 3). **P

    Techniques Used: Chromatin Immunoprecipitation, Cell Culture, Immunoprecipitation, Negative Control, Purification, Real-time Polymerase Chain Reaction, Sequencing, Amplification

    Effects of DEX on NeuroD1 promoter activity in AtT20 cells. (A) Effect of DEX on full-length NeuroD1 promoter activity. AtT20 cells transiently transfected with 300 ng m NeuroD1 -Luc (ND full: -2.2-kb/+150) and 150 ng pRSV-β-gal were treated with DEX (1 nM, 10 nM, or 100 nM) or 0.1% ethanol (vehicle control) for 24 hrs before the luciferase assay. Results are expressed as percentages of control (100%). Each point represents mean ± SEM (n = 4). ** P
    Figure Legend Snippet: Effects of DEX on NeuroD1 promoter activity in AtT20 cells. (A) Effect of DEX on full-length NeuroD1 promoter activity. AtT20 cells transiently transfected with 300 ng m NeuroD1 -Luc (ND full: -2.2-kb/+150) and 150 ng pRSV-β-gal were treated with DEX (1 nM, 10 nM, or 100 nM) or 0.1% ethanol (vehicle control) for 24 hrs before the luciferase assay. Results are expressed as percentages of control (100%). Each point represents mean ± SEM (n = 4). ** P

    Techniques Used: Activity Assay, Transfection, Luciferase

    Effects of NeuroD1 overexpression on Pomc mRNA expression and Pomc promoter activity in AtT20 cells. (A) Effect of NeuroD1 overexpression on the DEX-mediated Pomc mRNA decrease. AtT20 cells were transiently transfected with pcDNA3 and NeuroD1 plasmid (volume adjusted to 300 ng each with pcDNA3 empty vector) and incubated either in the presence (100 nM) or absence (control) of DEX for 24 hrs. Results are expressed as percentages of each control (100%). Each point represents mean ± SEM (n = 4). *** P
    Figure Legend Snippet: Effects of NeuroD1 overexpression on Pomc mRNA expression and Pomc promoter activity in AtT20 cells. (A) Effect of NeuroD1 overexpression on the DEX-mediated Pomc mRNA decrease. AtT20 cells were transiently transfected with pcDNA3 and NeuroD1 plasmid (volume adjusted to 300 ng each with pcDNA3 empty vector) and incubated either in the presence (100 nM) or absence (control) of DEX for 24 hrs. Results are expressed as percentages of each control (100%). Each point represents mean ± SEM (n = 4). *** P

    Techniques Used: Over Expression, Expressing, Activity Assay, Transfection, Plasmid Preparation, Incubation

    Effects of DEX on the mRNA expression of Tpit , Pitx1 , Nur77 and Nurr1 in AtT20 cells. (A) Time-dependent effect of DEX on Tpit mRNA expression. AtT20 cells were treated with DEX (100 nM) for 1 hr, 3 hrs, 6 hrs, 9 hrs, or 24 hrs. Vehicle control: 0.1% ethanol. Results are expressed as percentages of control (100%). Each point represents mean ± SEM (n = 4). NS denotes “Not Significant.” Two independent experiments were performed with consistent results. (B) Time-dependent effect of DEX on Pitx1 mRNA expression. AtT20 cells were treated with DEX (100 nM) for 1 hr, 3 hrs, 6 hrs, 9 hrs, or 24 hrs. Vehicle control: 0.1% ethanol. Results are expressed as percentages of each control (100%). NS denotes “Not Significant.” Two independent experiments were performed with consistent results. (C) Time-dependent effect of DEX on Nur77 mRNA expression. AtT20 cells were treated with DEX (100 nM) for 1 hr, 3 hrs, 6 hrs, 9 hrs, or 24 hrs. Vehicle control: 0.1% ethanol. Results are expressed as percentages of control (100%). NS denotes “Not Significant.” Two independent experiments were performed with consistent results. (D) Time-dependent effect of DEX on Nurr1 mRNA expression. AtT20 cells were treated with DEX (100 nM) for 1 hr, 3 hrs, 6 hrs, 9 hrs, or 24 hrs. Vehicle control: 0.1% ethanol. Results are expressed as percentages of control (100%). Each point represents mean ± SEM (n = 4). NS denotes “Not Significant.” *** P
    Figure Legend Snippet: Effects of DEX on the mRNA expression of Tpit , Pitx1 , Nur77 and Nurr1 in AtT20 cells. (A) Time-dependent effect of DEX on Tpit mRNA expression. AtT20 cells were treated with DEX (100 nM) for 1 hr, 3 hrs, 6 hrs, 9 hrs, or 24 hrs. Vehicle control: 0.1% ethanol. Results are expressed as percentages of control (100%). Each point represents mean ± SEM (n = 4). NS denotes “Not Significant.” Two independent experiments were performed with consistent results. (B) Time-dependent effect of DEX on Pitx1 mRNA expression. AtT20 cells were treated with DEX (100 nM) for 1 hr, 3 hrs, 6 hrs, 9 hrs, or 24 hrs. Vehicle control: 0.1% ethanol. Results are expressed as percentages of each control (100%). NS denotes “Not Significant.” Two independent experiments were performed with consistent results. (C) Time-dependent effect of DEX on Nur77 mRNA expression. AtT20 cells were treated with DEX (100 nM) for 1 hr, 3 hrs, 6 hrs, 9 hrs, or 24 hrs. Vehicle control: 0.1% ethanol. Results are expressed as percentages of control (100%). NS denotes “Not Significant.” Two independent experiments were performed with consistent results. (D) Time-dependent effect of DEX on Nurr1 mRNA expression. AtT20 cells were treated with DEX (100 nM) for 1 hr, 3 hrs, 6 hrs, 9 hrs, or 24 hrs. Vehicle control: 0.1% ethanol. Results are expressed as percentages of control (100%). Each point represents mean ± SEM (n = 4). NS denotes “Not Significant.” *** P

    Techniques Used: Expressing

    Effects of DEX on Pomc mRNA expression in AtT20 cells. (A) Dose-dependent effect of DEX on Pomc mRNA expression. AtT20 cells were treated with DEX (1 nM, 10 nM, or 100 nM) or 0.1% ethanol (vehicle control) for 24 hrs. Results are expressed as percentages of control (100%). Data represents mean ± SEM (n = 4). *** P
    Figure Legend Snippet: Effects of DEX on Pomc mRNA expression in AtT20 cells. (A) Dose-dependent effect of DEX on Pomc mRNA expression. AtT20 cells were treated with DEX (1 nM, 10 nM, or 100 nM) or 0.1% ethanol (vehicle control) for 24 hrs. Results are expressed as percentages of control (100%). Data represents mean ± SEM (n = 4). *** P

    Techniques Used: Expressing

    Effects of DEX on Pomc promoter activity in AtT20 cells. (A) Effect of DEX on full-length Pomc promoter activity. AtT20 cells transiently transfected with 300 ng full-length r Pomc -Luc (-703/+58-luc) and 100 ng pCMV-β-gal were treated with DEX (1 nM, 10 nM, or 100 nM) or 0.1% ethanol (vehicle control) for 24 hrs before the luciferase assay. Results are expressed as percentages of control (100%). Data represents mean ± SEM (n = 4). *** P
    Figure Legend Snippet: Effects of DEX on Pomc promoter activity in AtT20 cells. (A) Effect of DEX on full-length Pomc promoter activity. AtT20 cells transiently transfected with 300 ng full-length r Pomc -Luc (-703/+58-luc) and 100 ng pCMV-β-gal were treated with DEX (1 nM, 10 nM, or 100 nM) or 0.1% ethanol (vehicle control) for 24 hrs before the luciferase assay. Results are expressed as percentages of control (100%). Data represents mean ± SEM (n = 4). *** P

    Techniques Used: Activity Assay, Transfection, Luciferase

    Effects of DEX on the mRNA expression of NeuroD1 , Pan1 ( E47 ), and Rb in AtT20 cells. (A) Dose-dependent effect of DEX on NeuroD1 mRNA expression. AtT20 cells were treated with DEX (1 nM, 10 nM, or 100 nM) or 0.1% ethanol (vehicle control) for 24 hrs. Results are expressed as percentages of control (100%). Each point represents mean ± SEM (n = 4). *** P
    Figure Legend Snippet: Effects of DEX on the mRNA expression of NeuroD1 , Pan1 ( E47 ), and Rb in AtT20 cells. (A) Dose-dependent effect of DEX on NeuroD1 mRNA expression. AtT20 cells were treated with DEX (1 nM, 10 nM, or 100 nM) or 0.1% ethanol (vehicle control) for 24 hrs. Results are expressed as percentages of control (100%). Each point represents mean ± SEM (n = 4). *** P

    Techniques Used: Expressing

    25) Product Images from "Selective estrogen receptor modulators decrease invasiveness in pituitary adenoma cell lines AtT‐20 and TtT/GF by affecting expression of MMP‐14 and ADAM12"

    Article Title: Selective estrogen receptor modulators decrease invasiveness in pituitary adenoma cell lines AtT‐20 and TtT/GF by affecting expression of MMP‐14 and ADAM12

    Journal: FEBS Open Bio

    doi: 10.1002/2211-5463.12999

    Selective estrogen receptor modulators reduce invasiveness in PA cell lines AtT‐20 (B) and TtT/GF (C). (A) Representative images of AtT20 (upper panel) and TtT/GF (lower panel) cells stained with DAPI. Note that cell lines were treated with 25% inhibitory concentrations (IC 25 ) of SERMs bazedoxifene, clomiphene, and raloxifene as indicated in Table 1 . Scale bar in (A), for all images, 50 μm. Batimastat, a broad‐range MMP inhibitor, served as positive control. With bazedoxifene in TtT/GF cells, the outlier (o) was excluded from the statistical analysis. Values are given as median, first quartile, and third quartile. Values are obtained from three independent experiments performed in triplicates. t served as statistical test. *Statistically significant difference as compared to control.
    Figure Legend Snippet: Selective estrogen receptor modulators reduce invasiveness in PA cell lines AtT‐20 (B) and TtT/GF (C). (A) Representative images of AtT20 (upper panel) and TtT/GF (lower panel) cells stained with DAPI. Note that cell lines were treated with 25% inhibitory concentrations (IC 25 ) of SERMs bazedoxifene, clomiphene, and raloxifene as indicated in Table 1 . Scale bar in (A), for all images, 50 μm. Batimastat, a broad‐range MMP inhibitor, served as positive control. With bazedoxifene in TtT/GF cells, the outlier (o) was excluded from the statistical analysis. Values are given as median, first quartile, and third quartile. Values are obtained from three independent experiments performed in triplicates. t served as statistical test. *Statistically significant difference as compared to control.

    Techniques Used: Staining, Positive Control

    Transfection of AtT‐20 (A, B) and TtT/GF (C, D) cells with siRNA silencing ADAM12 and MMP‐14 genes and their effect on invasion. Western blots for the respective proteins in AtT‐20 (A) and TtT/GF (C) cells; scramble (‘scr’) siRNA served as transfection and tubulin as loading controls. Invasion rates after selective silencing of ADAM12 and MMP‐14 genes as compared to scramble siRNA in AtT‐20 (B) and TtT/GF cells (D) were calculated. Values are obtained from three independent experiments in triplicates and are indicated as median, first quartile, and third quartile. t served as statistical test. *Statistically significant difference as compared to control (Scramble).
    Figure Legend Snippet: Transfection of AtT‐20 (A, B) and TtT/GF (C, D) cells with siRNA silencing ADAM12 and MMP‐14 genes and their effect on invasion. Western blots for the respective proteins in AtT‐20 (A) and TtT/GF (C) cells; scramble (‘scr’) siRNA served as transfection and tubulin as loading controls. Invasion rates after selective silencing of ADAM12 and MMP‐14 genes as compared to scramble siRNA in AtT‐20 (B) and TtT/GF cells (D) were calculated. Values are obtained from three independent experiments in triplicates and are indicated as median, first quartile, and third quartile. t served as statistical test. *Statistically significant difference as compared to control (Scramble).

    Techniques Used: Transfection, Western Blot

    Western blot analyses of whole‐cell lysates from AtT‐20 cells (A, C) and TtT/GF cells (B, D) treated with 50% inhibitory concentrations (IC 50 , as indicated in Table 1 ) of SERMs bazedoxifene (B, red), clomiphene (C, green), and raloxifene (R, yellow) for three days. Protein levels of ADAM12 (A, B) and MMP‐14 (C, D) are shown from one representative experiment. Note that ADAM12 can be seen as 68‐kDa band (black arrowhead), whereas MMP‐14 is shown as a 66‐kDa band (black arrowhead) compared to solvent control (‘DMSO’). Blank served as an additional control to judge the vehicle effect. Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH, 35 kDa, white arrowhead) was used as loading control. Bands were quantified by imagej analysis from three quantifications. Values are given as mean ± SEM using one‐way ANOVA. t served as statistical test, with ** P
    Figure Legend Snippet: Western blot analyses of whole‐cell lysates from AtT‐20 cells (A, C) and TtT/GF cells (B, D) treated with 50% inhibitory concentrations (IC 50 , as indicated in Table 1 ) of SERMs bazedoxifene (B, red), clomiphene (C, green), and raloxifene (R, yellow) for three days. Protein levels of ADAM12 (A, B) and MMP‐14 (C, D) are shown from one representative experiment. Note that ADAM12 can be seen as 68‐kDa band (black arrowhead), whereas MMP‐14 is shown as a 66‐kDa band (black arrowhead) compared to solvent control (‘DMSO’). Blank served as an additional control to judge the vehicle effect. Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH, 35 kDa, white arrowhead) was used as loading control. Bands were quantified by imagej analysis from three quantifications. Values are given as mean ± SEM using one‐way ANOVA. t served as statistical test, with ** P

    Techniques Used: Western Blot

    Quantitative real‐time‐polymerase chain reaction to determine mRNA levels of ADAM12 in AtT‐20 (A), of Basigin in AtT‐20 cells (B), of ADAM12 in TtT/GF cells (C), of MMP‐14 in TtT/GF cells (D), and of basigin in TtT/GF cells (E) in response to SERM treatment. For each SERM (raloxifene, bazedoxifene, and clomiphene), 50% inhibitory concentrations (IC 50 ) as indicated in Table 1 were used to treat cells for 72 h. Quantitative PCR was performed in three independent experiments in triplicates. Values are given as median, first quartile, and third quartile. t served as statistical test. *Statistically significant difference as compared to control.
    Figure Legend Snippet: Quantitative real‐time‐polymerase chain reaction to determine mRNA levels of ADAM12 in AtT‐20 (A), of Basigin in AtT‐20 cells (B), of ADAM12 in TtT/GF cells (C), of MMP‐14 in TtT/GF cells (D), and of basigin in TtT/GF cells (E) in response to SERM treatment. For each SERM (raloxifene, bazedoxifene, and clomiphene), 50% inhibitory concentrations (IC 50 ) as indicated in Table 1 were used to treat cells for 72 h. Quantitative PCR was performed in three independent experiments in triplicates. Values are given as median, first quartile, and third quartile. t served as statistical test. *Statistically significant difference as compared to control.

    Techniques Used: Real-time Polymerase Chain Reaction

    Mean cycle number values as determined by qPCR representing absolute gene expression levels of MMP‐1, MMP‐9, MMP‐14, ADAM8, and ADAM12 in untreated PA cell lines AtT‐20 and TtT/GF.
    Figure Legend Snippet: Mean cycle number values as determined by qPCR representing absolute gene expression levels of MMP‐1, MMP‐9, MMP‐14, ADAM8, and ADAM12 in untreated PA cell lines AtT‐20 and TtT/GF.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    Dose–response curves for SERMs bazedoxifene, clomiphene, and raloxifene in PA cell lines AtT‐20 (A) and TtT/GF (B). Values are indicated as mean ± SEM. ctrl, control.
    Figure Legend Snippet: Dose–response curves for SERMs bazedoxifene, clomiphene, and raloxifene in PA cell lines AtT‐20 (A) and TtT/GF (B). Values are indicated as mean ± SEM. ctrl, control.

    Techniques Used:

    26) Product Images from "Hedgehog signaling in endocrine and folliculo-stellate cells of the adult pituitary"

    Article Title: Hedgehog signaling in endocrine and folliculo-stellate cells of the adult pituitary

    Journal: The Journal of Endocrinology

    doi: 10.1530/JOE-20-0388

    Paracrine signal transduction of Hh-activated FSC to somatotrophs is mediated by Vip/Vipr2 signaling. (A and B) qRT-PCR- (E, same samples as shown in Supplementary Fig. 5) and EIA-based (F) verification of increased Vip /Vip expression/secretion of SAG-treated TtT/GF cells (E) or supernatant (F, for total Vip concentration see Supplementary Fig. 8). (C, D and E) Vip mRNA (C), Vip protein (D) and Vipr2 mRNA expression of TtT/GF (C, D and E), GH3 and AtT-20 cells compared to NIH/3T3 cells (C and E). (F) qRT-PCR-based analysis of Gh expression level of GH3 cells treated with CM-TtT/GF SAG or CoM-TtT/GF supplemented with 1 µM Vip antagonist (VipAntag) or solvent, respectively. (G) Combined visualization of Gli1 transcripts and Vip protein expression in an adult murine C57Bl6/N wildtype pituitary gland. Gene expression levels in A, C, E and F were normalized to 18S rRNA expression and in A and F additionally to the Vip or Gh expression level of solvent-treated control cells, respectively (dotted lines in A and F). Vip protein concentration was normalized to the Vip protein concentration of solvent-treated control cells (dotted line in B). Each open circle indicates one biological replicate measured in technical triplicates. Mean ± s.e.m. Significant differences were tested using the non-parametric Holm–Sidak method. Significant differences to the respective base line (dotted lines) are indicated by asterisks above the data. * P = 0.05; ** P = 0.01; *** P = 0.001. White arrows: Vip + TtT/GF cells. White double arrows: Gli1 + Vip + stellate-shaped pituitary cells. Scale bars: 10 µm (D), 3.3 µm (G).
    Figure Legend Snippet: Paracrine signal transduction of Hh-activated FSC to somatotrophs is mediated by Vip/Vipr2 signaling. (A and B) qRT-PCR- (E, same samples as shown in Supplementary Fig. 5) and EIA-based (F) verification of increased Vip /Vip expression/secretion of SAG-treated TtT/GF cells (E) or supernatant (F, for total Vip concentration see Supplementary Fig. 8). (C, D and E) Vip mRNA (C), Vip protein (D) and Vipr2 mRNA expression of TtT/GF (C, D and E), GH3 and AtT-20 cells compared to NIH/3T3 cells (C and E). (F) qRT-PCR-based analysis of Gh expression level of GH3 cells treated with CM-TtT/GF SAG or CoM-TtT/GF supplemented with 1 µM Vip antagonist (VipAntag) or solvent, respectively. (G) Combined visualization of Gli1 transcripts and Vip protein expression in an adult murine C57Bl6/N wildtype pituitary gland. Gene expression levels in A, C, E and F were normalized to 18S rRNA expression and in A and F additionally to the Vip or Gh expression level of solvent-treated control cells, respectively (dotted lines in A and F). Vip protein concentration was normalized to the Vip protein concentration of solvent-treated control cells (dotted line in B). Each open circle indicates one biological replicate measured in technical triplicates. Mean ± s.e.m. Significant differences were tested using the non-parametric Holm–Sidak method. Significant differences to the respective base line (dotted lines) are indicated by asterisks above the data. * P = 0.05; ** P = 0.01; *** P = 0.001. White arrows: Vip + TtT/GF cells. White double arrows: Gli1 + Vip + stellate-shaped pituitary cells. Scale bars: 10 µm (D), 3.3 µm (G).

    Techniques Used: Transduction, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Concentration Assay, Protein Concentration

    27) Product Images from "Pterostilbene Decreases the Antioxidant Defenses of Aggressive Cancer Cells In Vivo: A Physiological Glucocorticoids- and Nrf2-Dependent Mechanism"

    Article Title: Pterostilbene Decreases the Antioxidant Defenses of Aggressive Cancer Cells In Vivo: A Physiological Glucocorticoids- and Nrf2-Dependent Mechanism

    Journal: Antioxidants & Redox Signaling

    doi: 10.1089/ars.2015.6437

    Pter bioavailability in brain and the pituitary gland and its effect on ACTH synthesis and POMC expression in AtT-20 cells. (A) Whole brain and pituitary gland levels of Pter after its i.v. administration (30 mg/kg) ( n = 6–7 mice; *Significantly different p
    Figure Legend Snippet: Pter bioavailability in brain and the pituitary gland and its effect on ACTH synthesis and POMC expression in AtT-20 cells. (A) Whole brain and pituitary gland levels of Pter after its i.v. administration (30 mg/kg) ( n = 6–7 mice; *Significantly different p

    Techniques Used: Expressing, Mouse Assay

    28) Product Images from "Identification of driver genes and key pathways of non-functional pituitary adenomas predicts the therapeutic effect of STO-609"

    Article Title: Identification of driver genes and key pathways of non-functional pituitary adenomas predicts the therapeutic effect of STO-609

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0240230

    (A) Cellular viability of HP75, GT1-1 and AtT-20 cells treated with STO-609 (B) Clonogenicities in Petri dishes with different doses of STO-609 (C) Scratch assay in control and STO-609 group (D) numbers of clone formation in HP75 cell line and GT1-1 cell line (E) Wound width in control and STO-609 (F) The distribution of cells in apoptosis with different doses of STO-609 (G) The percentage of apoptosis cell treated with different dose of STO-609.
    Figure Legend Snippet: (A) Cellular viability of HP75, GT1-1 and AtT-20 cells treated with STO-609 (B) Clonogenicities in Petri dishes with different doses of STO-609 (C) Scratch assay in control and STO-609 group (D) numbers of clone formation in HP75 cell line and GT1-1 cell line (E) Wound width in control and STO-609 (F) The distribution of cells in apoptosis with different doses of STO-609 (G) The percentage of apoptosis cell treated with different dose of STO-609.

    Techniques Used: Wound Healing Assay

    29) Product Images from "Region-Specific Regulation of RGS4 (Regulator of G-Protein–Signaling Protein Type 4) in Brain by Stress and Glucocorticoids: In Vivo and In VitroStudies"

    Article Title: Region-Specific Regulation of RGS4 (Regulator of G-Protein–Signaling Protein Type 4) in Brain by Stress and Glucocorticoids: In Vivo and In VitroStudies

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.19-10-03674.1999

    Effect of actinomycin D on dexamethasone regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 4).
    Figure Legend Snippet: Effect of actinomycin D on dexamethasone regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 4).

    Techniques Used: Expressing

    Time course study for dexamethasone regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 3–6; * p
    Figure Legend Snippet: Time course study for dexamethasone regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 3–6; * p

    Techniques Used: Expressing

    Dose–response analyses for dexamethasone ( Dex ) regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 3–6; * p
    Figure Legend Snippet: Dose–response analyses for dexamethasone ( Dex ) regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 3–6; * p

    Techniques Used: Expressing

    Comparison of the regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells by dexamethasone ( Dex ), CRF, and forskolin ( FK ). Data are expressed as the mean percent of control (± SEM; n = 4; * p
    Figure Legend Snippet: Comparison of the regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells by dexamethasone ( Dex ), CRF, and forskolin ( FK ). Data are expressed as the mean percent of control (± SEM; n = 4; * p

    Techniques Used: Expressing

    30) Product Images from "Region-Specific Regulation of RGS4 (Regulator of G-Protein–Signaling Protein Type 4) in Brain by Stress and Glucocorticoids: In Vivo and In VitroStudies"

    Article Title: Region-Specific Regulation of RGS4 (Regulator of G-Protein–Signaling Protein Type 4) in Brain by Stress and Glucocorticoids: In Vivo and In VitroStudies

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.19-10-03674.1999

    Effect of actinomycin D on dexamethasone regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 4).
    Figure Legend Snippet: Effect of actinomycin D on dexamethasone regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 4).

    Techniques Used: Expressing

    Time course study for dexamethasone regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 3–6; * p
    Figure Legend Snippet: Time course study for dexamethasone regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 3–6; * p

    Techniques Used: Expressing

    Dose–response analyses for dexamethasone ( Dex ) regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 3–6; * p
    Figure Legend Snippet: Dose–response analyses for dexamethasone ( Dex ) regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 3–6; * p

    Techniques Used: Expressing

    Comparison of the regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells by dexamethasone ( Dex ), CRF, and forskolin ( FK ). Data are expressed as the mean percent of control (± SEM; n = 4; * p
    Figure Legend Snippet: Comparison of the regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells by dexamethasone ( Dex ), CRF, and forskolin ( FK ). Data are expressed as the mean percent of control (± SEM; n = 4; * p

    Techniques Used: Expressing

    31) Product Images from "Functional characterization of DLK1/MEG3 locus on chromosome 14q32.2 reveals the differentiation of pituitary neuroendocrine tumors"

    Article Title: Functional characterization of DLK1/MEG3 locus on chromosome 14q32.2 reveals the differentiation of pituitary neuroendocrine tumors

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.202376

    Effect of anit-DLK1 antibody on the bioactivity of PitNET cell lines. ( A ) Western blot assay measured the levels of DLK1 and PIT1 in GH3 cell line, MMQ cell line and ATT20 cell line. ( B ) Anti-DLK1 antibody inhibited the cell viability of GH3 cells in the dose- and time-dependent manner, not MMQ cells or ATT20 cells. ( C ) Anti-DLK1 antibody inhibited the secretion of GH/IGF-1 in GH3 cells, not PRL in MMQ cells and ACTH in ATT20 cells. ( D ) Clone forming experiment showed the anti-DLK1 antibody promoted the cell proliferation in GH3 cell line. ( E ) Confocal experiment showed DLK1 regulated the level of PIT1 in GH3 cell line. ( F ) Western blot experiment showed Anti-DLK1 antibody activated the mTOR pathway in GH3 cell line. *compare to control group P
    Figure Legend Snippet: Effect of anit-DLK1 antibody on the bioactivity of PitNET cell lines. ( A ) Western blot assay measured the levels of DLK1 and PIT1 in GH3 cell line, MMQ cell line and ATT20 cell line. ( B ) Anti-DLK1 antibody inhibited the cell viability of GH3 cells in the dose- and time-dependent manner, not MMQ cells or ATT20 cells. ( C ) Anti-DLK1 antibody inhibited the secretion of GH/IGF-1 in GH3 cells, not PRL in MMQ cells and ACTH in ATT20 cells. ( D ) Clone forming experiment showed the anti-DLK1 antibody promoted the cell proliferation in GH3 cell line. ( E ) Confocal experiment showed DLK1 regulated the level of PIT1 in GH3 cell line. ( F ) Western blot experiment showed Anti-DLK1 antibody activated the mTOR pathway in GH3 cell line. *compare to control group P

    Techniques Used: Western Blot

    Effect of siMEG3 on the bioactivity of PitNET cell lines. ( A ) RT-qPCR experiment measured the RNAi efficiency of siMEG3-1 and siMEG3-2. ( B ) Western blot experiment showed RNAi-MEG3 reduced the level of DLK1, PIT1 and GH in GH3 cell line. ( C ) RNAi-MEG3 obviously increased the cell viability of GH3 cell line, mildly increase in MMQ cell line, and no change in ATT20 cell line. ( D ) RNAi-MEG3 inhibited the secretion of GH/IGF-1 in GH3 cells, not PRL in MMQ cells and ACTH in ATT20 cells. ( E ) Confocal experiment showed RNAi-MEG3 inhibited the levels of DLK1 and PIT1 in GH3 cells. *compare to control group P
    Figure Legend Snippet: Effect of siMEG3 on the bioactivity of PitNET cell lines. ( A ) RT-qPCR experiment measured the RNAi efficiency of siMEG3-1 and siMEG3-2. ( B ) Western blot experiment showed RNAi-MEG3 reduced the level of DLK1, PIT1 and GH in GH3 cell line. ( C ) RNAi-MEG3 obviously increased the cell viability of GH3 cell line, mildly increase in MMQ cell line, and no change in ATT20 cell line. ( D ) RNAi-MEG3 inhibited the secretion of GH/IGF-1 in GH3 cells, not PRL in MMQ cells and ACTH in ATT20 cells. ( E ) Confocal experiment showed RNAi-MEG3 inhibited the levels of DLK1 and PIT1 in GH3 cells. *compare to control group P

    Techniques Used: Quantitative RT-PCR, Western Blot

    32) Product Images from "miR-15a/miR-16-1 expression inversely correlates with cyclin D1 levels in Men1 pituitary NETs"

    Article Title: miR-15a/miR-16-1 expression inversely correlates with cyclin D1 levels in Men1 pituitary NETs

    Journal: The Journal of Endocrinology

    doi: 10.1530/JOE-18-0278

    Relationship between miR-15a/miR-16-1 and menin in HeLa and AtT20 cells. Significant differences were not observed in the levels of Men1 or menin expression after antagomir treatment of HeLa cells, evaluated by qRT-PCR (A) and Western blot (B), respectively. Use of MEN1 -specific siRNA in HeLa and AtT20 cells decreased expression of MEN1 (C) (assessed by qRT-PCR, *** P
    Figure Legend Snippet: Relationship between miR-15a/miR-16-1 and menin in HeLa and AtT20 cells. Significant differences were not observed in the levels of Men1 or menin expression after antagomir treatment of HeLa cells, evaluated by qRT-PCR (A) and Western blot (B), respectively. Use of MEN1 -specific siRNA in HeLa and AtT20 cells decreased expression of MEN1 (C) (assessed by qRT-PCR, *** P

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot

    33) Product Images from "Selective estrogen receptor modulators decrease invasiveness in pituitary adenoma cell lines AtT‐20 and TtT/GF by affecting expression of MMP‐14 and ADAM12"

    Article Title: Selective estrogen receptor modulators decrease invasiveness in pituitary adenoma cell lines AtT‐20 and TtT/GF by affecting expression of MMP‐14 and ADAM12

    Journal: FEBS Open Bio

    doi: 10.1002/2211-5463.12999

    Selective estrogen receptor modulators reduce invasiveness in PA cell lines AtT‐20 (B) and TtT/GF (C). (A) Representative images of AtT20 (upper panel) and TtT/GF (lower panel) cells stained with DAPI. Note that cell lines were treated with 25% inhibitory concentrations (IC 25 ) of SERMs bazedoxifene, clomiphene, and raloxifene as indicated in Table 1 . Scale bar in (A), for all images, 50 μm. Batimastat, a broad‐range MMP inhibitor, served as positive control. With bazedoxifene in TtT/GF cells, the outlier (o) was excluded from the statistical analysis. Values are given as median, first quartile, and third quartile. Values are obtained from three independent experiments performed in triplicates. t served as statistical test. *Statistically significant difference as compared to control.
    Figure Legend Snippet: Selective estrogen receptor modulators reduce invasiveness in PA cell lines AtT‐20 (B) and TtT/GF (C). (A) Representative images of AtT20 (upper panel) and TtT/GF (lower panel) cells stained with DAPI. Note that cell lines were treated with 25% inhibitory concentrations (IC 25 ) of SERMs bazedoxifene, clomiphene, and raloxifene as indicated in Table 1 . Scale bar in (A), for all images, 50 μm. Batimastat, a broad‐range MMP inhibitor, served as positive control. With bazedoxifene in TtT/GF cells, the outlier (o) was excluded from the statistical analysis. Values are given as median, first quartile, and third quartile. Values are obtained from three independent experiments performed in triplicates. t served as statistical test. *Statistically significant difference as compared to control.

    Techniques Used: Staining, Positive Control

    Transfection of AtT‐20 (A, B) and TtT/GF (C, D) cells with siRNA silencing ADAM12 and MMP‐14 genes and their effect on invasion. Western blots for the respective proteins in AtT‐20 (A) and TtT/GF (C) cells; scramble (‘scr’) siRNA served as transfection and tubulin as loading controls. Invasion rates after selective silencing of ADAM12 and MMP‐14 genes as compared to scramble siRNA in AtT‐20 (B) and TtT/GF cells (D) were calculated. Values are obtained from three independent experiments in triplicates and are indicated as median, first quartile, and third quartile. t served as statistical test. *Statistically significant difference as compared to control (Scramble).
    Figure Legend Snippet: Transfection of AtT‐20 (A, B) and TtT/GF (C, D) cells with siRNA silencing ADAM12 and MMP‐14 genes and their effect on invasion. Western blots for the respective proteins in AtT‐20 (A) and TtT/GF (C) cells; scramble (‘scr’) siRNA served as transfection and tubulin as loading controls. Invasion rates after selective silencing of ADAM12 and MMP‐14 genes as compared to scramble siRNA in AtT‐20 (B) and TtT/GF cells (D) were calculated. Values are obtained from three independent experiments in triplicates and are indicated as median, first quartile, and third quartile. t served as statistical test. *Statistically significant difference as compared to control (Scramble).

    Techniques Used: Transfection, Western Blot

    Western blot analyses of whole‐cell lysates from AtT‐20 cells (A, C) and TtT/GF cells (B, D) treated with 50% inhibitory concentrations (IC 50 , as indicated in Table 1 ) of SERMs bazedoxifene (B, red), clomiphene (C, green), and raloxifene (R, yellow) for three days. Protein levels of ADAM12 (A, B) and MMP‐14 (C, D) are shown from one representative experiment. Note that ADAM12 can be seen as 68‐kDa band (black arrowhead), whereas MMP‐14 is shown as a 66‐kDa band (black arrowhead) compared to solvent control (‘DMSO’). Blank served as an additional control to judge the vehicle effect. Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH, 35 kDa, white arrowhead) was used as loading control. Bands were quantified by imagej analysis from three quantifications. Values are given as mean ± SEM using one‐way ANOVA. t served as statistical test, with ** P
    Figure Legend Snippet: Western blot analyses of whole‐cell lysates from AtT‐20 cells (A, C) and TtT/GF cells (B, D) treated with 50% inhibitory concentrations (IC 50 , as indicated in Table 1 ) of SERMs bazedoxifene (B, red), clomiphene (C, green), and raloxifene (R, yellow) for three days. Protein levels of ADAM12 (A, B) and MMP‐14 (C, D) are shown from one representative experiment. Note that ADAM12 can be seen as 68‐kDa band (black arrowhead), whereas MMP‐14 is shown as a 66‐kDa band (black arrowhead) compared to solvent control (‘DMSO’). Blank served as an additional control to judge the vehicle effect. Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH, 35 kDa, white arrowhead) was used as loading control. Bands were quantified by imagej analysis from three quantifications. Values are given as mean ± SEM using one‐way ANOVA. t served as statistical test, with ** P

    Techniques Used: Western Blot

    Quantitative real‐time‐polymerase chain reaction to determine mRNA levels of ADAM12 in AtT‐20 (A), of Basigin in AtT‐20 cells (B), of ADAM12 in TtT/GF cells (C), of MMP‐14 in TtT/GF cells (D), and of basigin in TtT/GF cells (E) in response to SERM treatment. For each SERM (raloxifene, bazedoxifene, and clomiphene), 50% inhibitory concentrations (IC 50 ) as indicated in Table 1 were used to treat cells for 72 h. Quantitative PCR was performed in three independent experiments in triplicates. Values are given as median, first quartile, and third quartile. t served as statistical test. *Statistically significant difference as compared to control.
    Figure Legend Snippet: Quantitative real‐time‐polymerase chain reaction to determine mRNA levels of ADAM12 in AtT‐20 (A), of Basigin in AtT‐20 cells (B), of ADAM12 in TtT/GF cells (C), of MMP‐14 in TtT/GF cells (D), and of basigin in TtT/GF cells (E) in response to SERM treatment. For each SERM (raloxifene, bazedoxifene, and clomiphene), 50% inhibitory concentrations (IC 50 ) as indicated in Table 1 were used to treat cells for 72 h. Quantitative PCR was performed in three independent experiments in triplicates. Values are given as median, first quartile, and third quartile. t served as statistical test. *Statistically significant difference as compared to control.

    Techniques Used: Real-time Polymerase Chain Reaction

    Mean cycle number values as determined by qPCR representing absolute gene expression levels of MMP‐1, MMP‐9, MMP‐14, ADAM8, and ADAM12 in untreated PA cell lines AtT‐20 and TtT/GF.
    Figure Legend Snippet: Mean cycle number values as determined by qPCR representing absolute gene expression levels of MMP‐1, MMP‐9, MMP‐14, ADAM8, and ADAM12 in untreated PA cell lines AtT‐20 and TtT/GF.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    Dose–response curves for SERMs bazedoxifene, clomiphene, and raloxifene in PA cell lines AtT‐20 (A) and TtT/GF (B). Values are indicated as mean ± SEM. ctrl, control.
    Figure Legend Snippet: Dose–response curves for SERMs bazedoxifene, clomiphene, and raloxifene in PA cell lines AtT‐20 (A) and TtT/GF (B). Values are indicated as mean ± SEM. ctrl, control.

    Techniques Used:

    34) Product Images from "Selective estrogen receptor modulators decrease invasiveness in pituitary adenoma cell lines AtT‐20 and TtT/GF by affecting expression of MMP‐14 and ADAM12"

    Article Title: Selective estrogen receptor modulators decrease invasiveness in pituitary adenoma cell lines AtT‐20 and TtT/GF by affecting expression of MMP‐14 and ADAM12

    Journal: FEBS Open Bio

    doi: 10.1002/2211-5463.12999

    Selective estrogen receptor modulators reduce invasiveness in PA cell lines AtT‐20 (B) and TtT/GF (C). (A) Representative images of AtT20 (upper panel) and TtT/GF (lower panel) cells stained with DAPI. Note that cell lines were treated with 25% inhibitory concentrations (IC 25 ) of SERMs bazedoxifene, clomiphene, and raloxifene as indicated in Table 1 . Scale bar in (A), for all images, 50 μm. Batimastat, a broad‐range MMP inhibitor, served as positive control. With bazedoxifene in TtT/GF cells, the outlier (o) was excluded from the statistical analysis. Values are given as median, first quartile, and third quartile. Values are obtained from three independent experiments performed in triplicates. t served as statistical test. *Statistically significant difference as compared to control.
    Figure Legend Snippet: Selective estrogen receptor modulators reduce invasiveness in PA cell lines AtT‐20 (B) and TtT/GF (C). (A) Representative images of AtT20 (upper panel) and TtT/GF (lower panel) cells stained with DAPI. Note that cell lines were treated with 25% inhibitory concentrations (IC 25 ) of SERMs bazedoxifene, clomiphene, and raloxifene as indicated in Table 1 . Scale bar in (A), for all images, 50 μm. Batimastat, a broad‐range MMP inhibitor, served as positive control. With bazedoxifene in TtT/GF cells, the outlier (o) was excluded from the statistical analysis. Values are given as median, first quartile, and third quartile. Values are obtained from three independent experiments performed in triplicates. t served as statistical test. *Statistically significant difference as compared to control.

    Techniques Used: Staining, Positive Control

    Transfection of AtT‐20 (A, B) and TtT/GF (C, D) cells with siRNA silencing ADAM12 and MMP‐14 genes and their effect on invasion. Western blots for the respective proteins in AtT‐20 (A) and TtT/GF (C) cells; scramble (‘scr’) siRNA served as transfection and tubulin as loading controls. Invasion rates after selective silencing of ADAM12 and MMP‐14 genes as compared to scramble siRNA in AtT‐20 (B) and TtT/GF cells (D) were calculated. Values are obtained from three independent experiments in triplicates and are indicated as median, first quartile, and third quartile. t served as statistical test. *Statistically significant difference as compared to control (Scramble).
    Figure Legend Snippet: Transfection of AtT‐20 (A, B) and TtT/GF (C, D) cells with siRNA silencing ADAM12 and MMP‐14 genes and their effect on invasion. Western blots for the respective proteins in AtT‐20 (A) and TtT/GF (C) cells; scramble (‘scr’) siRNA served as transfection and tubulin as loading controls. Invasion rates after selective silencing of ADAM12 and MMP‐14 genes as compared to scramble siRNA in AtT‐20 (B) and TtT/GF cells (D) were calculated. Values are obtained from three independent experiments in triplicates and are indicated as median, first quartile, and third quartile. t served as statistical test. *Statistically significant difference as compared to control (Scramble).

    Techniques Used: Transfection, Western Blot

    Western blot analyses of whole‐cell lysates from AtT‐20 cells (A, C) and TtT/GF cells (B, D) treated with 50% inhibitory concentrations (IC 50 , as indicated in Table 1 ) of SERMs bazedoxifene (B, red), clomiphene (C, green), and raloxifene (R, yellow) for three days. Protein levels of ADAM12 (A, B) and MMP‐14 (C, D) are shown from one representative experiment. Note that ADAM12 can be seen as 68‐kDa band (black arrowhead), whereas MMP‐14 is shown as a 66‐kDa band (black arrowhead) compared to solvent control (‘DMSO’). Blank served as an additional control to judge the vehicle effect. Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH, 35 kDa, white arrowhead) was used as loading control. Bands were quantified by imagej analysis from three quantifications. Values are given as mean ± SEM using one‐way ANOVA. t served as statistical test, with ** P
    Figure Legend Snippet: Western blot analyses of whole‐cell lysates from AtT‐20 cells (A, C) and TtT/GF cells (B, D) treated with 50% inhibitory concentrations (IC 50 , as indicated in Table 1 ) of SERMs bazedoxifene (B, red), clomiphene (C, green), and raloxifene (R, yellow) for three days. Protein levels of ADAM12 (A, B) and MMP‐14 (C, D) are shown from one representative experiment. Note that ADAM12 can be seen as 68‐kDa band (black arrowhead), whereas MMP‐14 is shown as a 66‐kDa band (black arrowhead) compared to solvent control (‘DMSO’). Blank served as an additional control to judge the vehicle effect. Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH, 35 kDa, white arrowhead) was used as loading control. Bands were quantified by imagej analysis from three quantifications. Values are given as mean ± SEM using one‐way ANOVA. t served as statistical test, with ** P

    Techniques Used: Western Blot

    Quantitative real‐time‐polymerase chain reaction to determine mRNA levels of ADAM12 in AtT‐20 (A), of Basigin in AtT‐20 cells (B), of ADAM12 in TtT/GF cells (C), of MMP‐14 in TtT/GF cells (D), and of basigin in TtT/GF cells (E) in response to SERM treatment. For each SERM (raloxifene, bazedoxifene, and clomiphene), 50% inhibitory concentrations (IC 50 ) as indicated in Table 1 were used to treat cells for 72 h. Quantitative PCR was performed in three independent experiments in triplicates. Values are given as median, first quartile, and third quartile. t served as statistical test. *Statistically significant difference as compared to control.
    Figure Legend Snippet: Quantitative real‐time‐polymerase chain reaction to determine mRNA levels of ADAM12 in AtT‐20 (A), of Basigin in AtT‐20 cells (B), of ADAM12 in TtT/GF cells (C), of MMP‐14 in TtT/GF cells (D), and of basigin in TtT/GF cells (E) in response to SERM treatment. For each SERM (raloxifene, bazedoxifene, and clomiphene), 50% inhibitory concentrations (IC 50 ) as indicated in Table 1 were used to treat cells for 72 h. Quantitative PCR was performed in three independent experiments in triplicates. Values are given as median, first quartile, and third quartile. t served as statistical test. *Statistically significant difference as compared to control.

    Techniques Used: Real-time Polymerase Chain Reaction

    Mean cycle number values as determined by qPCR representing absolute gene expression levels of MMP‐1, MMP‐9, MMP‐14, ADAM8, and ADAM12 in untreated PA cell lines AtT‐20 and TtT/GF.
    Figure Legend Snippet: Mean cycle number values as determined by qPCR representing absolute gene expression levels of MMP‐1, MMP‐9, MMP‐14, ADAM8, and ADAM12 in untreated PA cell lines AtT‐20 and TtT/GF.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    Dose–response curves for SERMs bazedoxifene, clomiphene, and raloxifene in PA cell lines AtT‐20 (A) and TtT/GF (B). Values are indicated as mean ± SEM. ctrl, control.
    Figure Legend Snippet: Dose–response curves for SERMs bazedoxifene, clomiphene, and raloxifene in PA cell lines AtT‐20 (A) and TtT/GF (B). Values are indicated as mean ± SEM. ctrl, control.

    Techniques Used:

    35) Product Images from "Selective estrogen receptor modulators decrease invasiveness in pituitary adenoma cell lines AtT‐20 and TtT/GF by affecting expression of MMP‐14 and ADAM12"

    Article Title: Selective estrogen receptor modulators decrease invasiveness in pituitary adenoma cell lines AtT‐20 and TtT/GF by affecting expression of MMP‐14 and ADAM12

    Journal: FEBS Open Bio

    doi: 10.1002/2211-5463.12999

    Selective estrogen receptor modulators reduce invasiveness in PA cell lines AtT‐20 (B) and TtT/GF (C). (A) Representative images of AtT20 (upper panel) and TtT/GF (lower panel) cells stained with DAPI. Note that cell lines were treated with 25% inhibitory concentrations (IC 25 ) of SERMs bazedoxifene, clomiphene, and raloxifene as indicated in Table 1 . Scale bar in (A), for all images, 50 μm. Batimastat, a broad‐range MMP inhibitor, served as positive control. With bazedoxifene in TtT/GF cells, the outlier (o) was excluded from the statistical analysis. Values are given as median, first quartile, and third quartile. Values are obtained from three independent experiments performed in triplicates. t served as statistical test. *Statistically significant difference as compared to control.
    Figure Legend Snippet: Selective estrogen receptor modulators reduce invasiveness in PA cell lines AtT‐20 (B) and TtT/GF (C). (A) Representative images of AtT20 (upper panel) and TtT/GF (lower panel) cells stained with DAPI. Note that cell lines were treated with 25% inhibitory concentrations (IC 25 ) of SERMs bazedoxifene, clomiphene, and raloxifene as indicated in Table 1 . Scale bar in (A), for all images, 50 μm. Batimastat, a broad‐range MMP inhibitor, served as positive control. With bazedoxifene in TtT/GF cells, the outlier (o) was excluded from the statistical analysis. Values are given as median, first quartile, and third quartile. Values are obtained from three independent experiments performed in triplicates. t served as statistical test. *Statistically significant difference as compared to control.

    Techniques Used: Staining, Positive Control

    Transfection of AtT‐20 (A, B) and TtT/GF (C, D) cells with siRNA silencing ADAM12 and MMP‐14 genes and their effect on invasion. Western blots for the respective proteins in AtT‐20 (A) and TtT/GF (C) cells; scramble (‘scr’) siRNA served as transfection and tubulin as loading controls. Invasion rates after selective silencing of ADAM12 and MMP‐14 genes as compared to scramble siRNA in AtT‐20 (B) and TtT/GF cells (D) were calculated. Values are obtained from three independent experiments in triplicates and are indicated as median, first quartile, and third quartile. t served as statistical test. *Statistically significant difference as compared to control (Scramble).
    Figure Legend Snippet: Transfection of AtT‐20 (A, B) and TtT/GF (C, D) cells with siRNA silencing ADAM12 and MMP‐14 genes and their effect on invasion. Western blots for the respective proteins in AtT‐20 (A) and TtT/GF (C) cells; scramble (‘scr’) siRNA served as transfection and tubulin as loading controls. Invasion rates after selective silencing of ADAM12 and MMP‐14 genes as compared to scramble siRNA in AtT‐20 (B) and TtT/GF cells (D) were calculated. Values are obtained from three independent experiments in triplicates and are indicated as median, first quartile, and third quartile. t served as statistical test. *Statistically significant difference as compared to control (Scramble).

    Techniques Used: Transfection, Western Blot

    Western blot analyses of whole‐cell lysates from AtT‐20 cells (A, C) and TtT/GF cells (B, D) treated with 50% inhibitory concentrations (IC 50 , as indicated in Table 1 ) of SERMs bazedoxifene (B, red), clomiphene (C, green), and raloxifene (R, yellow) for three days. Protein levels of ADAM12 (A, B) and MMP‐14 (C, D) are shown from one representative experiment. Note that ADAM12 can be seen as 68‐kDa band (black arrowhead), whereas MMP‐14 is shown as a 66‐kDa band (black arrowhead) compared to solvent control (‘DMSO’). Blank served as an additional control to judge the vehicle effect. Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH, 35 kDa, white arrowhead) was used as loading control. Bands were quantified by imagej analysis from three quantifications. Values are given as mean ± SEM using one‐way ANOVA. t served as statistical test, with ** P
    Figure Legend Snippet: Western blot analyses of whole‐cell lysates from AtT‐20 cells (A, C) and TtT/GF cells (B, D) treated with 50% inhibitory concentrations (IC 50 , as indicated in Table 1 ) of SERMs bazedoxifene (B, red), clomiphene (C, green), and raloxifene (R, yellow) for three days. Protein levels of ADAM12 (A, B) and MMP‐14 (C, D) are shown from one representative experiment. Note that ADAM12 can be seen as 68‐kDa band (black arrowhead), whereas MMP‐14 is shown as a 66‐kDa band (black arrowhead) compared to solvent control (‘DMSO’). Blank served as an additional control to judge the vehicle effect. Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH, 35 kDa, white arrowhead) was used as loading control. Bands were quantified by imagej analysis from three quantifications. Values are given as mean ± SEM using one‐way ANOVA. t served as statistical test, with ** P

    Techniques Used: Western Blot

    Quantitative real‐time‐polymerase chain reaction to determine mRNA levels of ADAM12 in AtT‐20 (A), of Basigin in AtT‐20 cells (B), of ADAM12 in TtT/GF cells (C), of MMP‐14 in TtT/GF cells (D), and of basigin in TtT/GF cells (E) in response to SERM treatment. For each SERM (raloxifene, bazedoxifene, and clomiphene), 50% inhibitory concentrations (IC 50 ) as indicated in Table 1 were used to treat cells for 72 h. Quantitative PCR was performed in three independent experiments in triplicates. Values are given as median, first quartile, and third quartile. t served as statistical test. *Statistically significant difference as compared to control.
    Figure Legend Snippet: Quantitative real‐time‐polymerase chain reaction to determine mRNA levels of ADAM12 in AtT‐20 (A), of Basigin in AtT‐20 cells (B), of ADAM12 in TtT/GF cells (C), of MMP‐14 in TtT/GF cells (D), and of basigin in TtT/GF cells (E) in response to SERM treatment. For each SERM (raloxifene, bazedoxifene, and clomiphene), 50% inhibitory concentrations (IC 50 ) as indicated in Table 1 were used to treat cells for 72 h. Quantitative PCR was performed in three independent experiments in triplicates. Values are given as median, first quartile, and third quartile. t served as statistical test. *Statistically significant difference as compared to control.

    Techniques Used: Real-time Polymerase Chain Reaction

    Mean cycle number values as determined by qPCR representing absolute gene expression levels of MMP‐1, MMP‐9, MMP‐14, ADAM8, and ADAM12 in untreated PA cell lines AtT‐20 and TtT/GF.
    Figure Legend Snippet: Mean cycle number values as determined by qPCR representing absolute gene expression levels of MMP‐1, MMP‐9, MMP‐14, ADAM8, and ADAM12 in untreated PA cell lines AtT‐20 and TtT/GF.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    Dose–response curves for SERMs bazedoxifene, clomiphene, and raloxifene in PA cell lines AtT‐20 (A) and TtT/GF (B). Values are indicated as mean ± SEM. ctrl, control.
    Figure Legend Snippet: Dose–response curves for SERMs bazedoxifene, clomiphene, and raloxifene in PA cell lines AtT‐20 (A) and TtT/GF (B). Values are indicated as mean ± SEM. ctrl, control.

    Techniques Used:

    36) Product Images from "Angpt2/Tie2 autostimulatory loop controls tumorigenesis"

    Article Title: Angpt2/Tie2 autostimulatory loop controls tumorigenesis

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.202114364

    Tie2 on PitNET cells is phosphorylated and can be stimulated by Angpt2 Co‐IF for both P‐Tie2 (Tyr 1102/ 1108; red) and for Na + K + ATPase (green), used as plasma membrane marker, of GH3 cells transduced with unspecific shRNA (shCtrl) and sh Angpt2 (#2) and incubated with rhANGPT2 or left untreated. Original magnification: 400×; scale bar: 20 μm. Quantification of P‐Tie2 immunostaining intensity in cells shown in (A) shCtrl‐transduced GH3 cells (266.71 ± 12.12); in sh Angpt2 cells (176.04 ± 8.46); in control cells treated with rhANGPT2: shCtrl + rhANGPT2: 246.33 ± 9.79 (versus shCtrl, not significant by t ‐test); in treated knockdown cells: sh Angpt2 + rhANGPT2: 218.68 ± 18.16 (versus shC2; * P = 0.0421 by t ‐test). The experiment was performed twice each with three technical replicates. Intensities are expressed as arbitrary units ± SEM. *** P
    Figure Legend Snippet: Tie2 on PitNET cells is phosphorylated and can be stimulated by Angpt2 Co‐IF for both P‐Tie2 (Tyr 1102/ 1108; red) and for Na + K + ATPase (green), used as plasma membrane marker, of GH3 cells transduced with unspecific shRNA (shCtrl) and sh Angpt2 (#2) and incubated with rhANGPT2 or left untreated. Original magnification: 400×; scale bar: 20 μm. Quantification of P‐Tie2 immunostaining intensity in cells shown in (A) shCtrl‐transduced GH3 cells (266.71 ± 12.12); in sh Angpt2 cells (176.04 ± 8.46); in control cells treated with rhANGPT2: shCtrl + rhANGPT2: 246.33 ± 9.79 (versus shCtrl, not significant by t ‐test); in treated knockdown cells: sh Angpt2 + rhANGPT2: 218.68 ± 18.16 (versus shC2; * P = 0.0421 by t ‐test). The experiment was performed twice each with three technical replicates. Intensities are expressed as arbitrary units ± SEM. *** P

    Techniques Used: Marker, Transduction, shRNA, Incubation, Immunostaining

    Tie2 on PitNET cells interacts with Angpt2 Proximity ligation assay (PLA) was performed on FFPE sections of rat PitNETs ( n = 5) using antibodies against both Angpt2 and Tie2. Nuclei were counterstained with DAPI (blue). Original magnification: 400×; scale bar: 20 µm. Quantification of the interactions between Angpt2 and Tie2 in rat PitNET tissues versus negative controls obtained using only 1 antibody (Appendix Fig S7 C). Box plots show 25 th to 75 th percentiles (box) and 5 th to 95 th percentiles (whiskers). The line in the box represents the median, whereas the “x” represents the mean. PLA was performed as in (A) on isolated rat primary NF‐PitNET stimulated with rhANGPT2. Quantification of Angpt2/Tie2 interactions in primary NF‐PitNET incubated with/without rhANGPT2. The total number of cells counted is reported in parenthesis (i.e., 212 and 345) as well as the number of positive cells (= showing at least one interaction). The graphs show the percentage of cells having the number of interactions reported on the x axis. Box plots show 25 th to 75 th percentiles (box) and 5 th to 95 th percentiles (whiskers). The line in the box represents the median, the “x” represents the mean, and the circle outlier points. Data information: (C, D) Pictures were taken with the same exposure time. Results shown are representative of the stainings results across all samples ( n = 4, n = 3, respectively). Source data are available online for this figure.
    Figure Legend Snippet: Tie2 on PitNET cells interacts with Angpt2 Proximity ligation assay (PLA) was performed on FFPE sections of rat PitNETs ( n = 5) using antibodies against both Angpt2 and Tie2. Nuclei were counterstained with DAPI (blue). Original magnification: 400×; scale bar: 20 µm. Quantification of the interactions between Angpt2 and Tie2 in rat PitNET tissues versus negative controls obtained using only 1 antibody (Appendix Fig S7 C). Box plots show 25 th to 75 th percentiles (box) and 5 th to 95 th percentiles (whiskers). The line in the box represents the median, whereas the “x” represents the mean. PLA was performed as in (A) on isolated rat primary NF‐PitNET stimulated with rhANGPT2. Quantification of Angpt2/Tie2 interactions in primary NF‐PitNET incubated with/without rhANGPT2. The total number of cells counted is reported in parenthesis (i.e., 212 and 345) as well as the number of positive cells (= showing at least one interaction). The graphs show the percentage of cells having the number of interactions reported on the x axis. Box plots show 25 th to 75 th percentiles (box) and 5 th to 95 th percentiles (whiskers). The line in the box represents the median, the “x” represents the mean, and the circle outlier points. Data information: (C, D) Pictures were taken with the same exposure time. Results shown are representative of the stainings results across all samples ( n = 4, n = 3, respectively). Source data are available online for this figure.

    Techniques Used: Proximity Ligation Assay, Formalin-fixed Paraffin-Embedded, Isolation, Incubation

    Pharmacological inhibition of Ang/Tie2 signaling suppresses growth and induces apoptosis of PitNET cells xenograft models and of autochthonous PitNETs in MENX rats in vivo Scheme of the in vivo treatment of GH3‐derived xenografts with AMG386. T2‐weighted images of two xenografted tumors taken at day 0 and 21 (largest diameter). The tumors are representative of the two treatment groups Scale bar: 2 mm. Changes in tumor volumes as determined by MRI were normalized to the day 0 value (= 100%) for each animal. All box plots show 25 th to 75 th percentiles (box) and 5 th to 95 th percentiles (whiskers). The line in the box represents the median. ** P = 0.007 (one way ANOVA). Changes in tumor volumes in female rats treated with AMG386 ( n = 6) or with placebo ( n = 5) for 14 days as determined by anatomical MRI, normalized to the day 0 value. Box plots show 25 th to 75 th percentiles (box) and 5 th to 95 th percentiles (whiskers). The line in the box represents the median. P = 0.0673, not significant (one way ANOVA). ADC values of the rat PitNETs before (day 0) and after (day 14) treatment with AMG386 (red, n = 8) or placebo (blue, n = 5). Box plots show 25 th to 75 th percentiles (box) and 5 th to 95 th percentiles (whiskers). The line in the box represents the median. ** P = 0.009; n.s., not significant ( t ‐test). Ex vivo expression of Tie2, P‐Tie2, Ki67 and Annexin V in PitNETs of female rats treated with AMG386 or placebo for 14 days. Original magnification: 200×; scale bar: 50 µm. Panels shown are representative of the two animal groups. Quantification of Annexin V intensity from tissues stained as in (F) ( n = 4/group). Three different areas per tumor sample were analyzed. Intensities are expressed as arbitrary units ± SEM. *** P
    Figure Legend Snippet: Pharmacological inhibition of Ang/Tie2 signaling suppresses growth and induces apoptosis of PitNET cells xenograft models and of autochthonous PitNETs in MENX rats in vivo Scheme of the in vivo treatment of GH3‐derived xenografts with AMG386. T2‐weighted images of two xenografted tumors taken at day 0 and 21 (largest diameter). The tumors are representative of the two treatment groups Scale bar: 2 mm. Changes in tumor volumes as determined by MRI were normalized to the day 0 value (= 100%) for each animal. All box plots show 25 th to 75 th percentiles (box) and 5 th to 95 th percentiles (whiskers). The line in the box represents the median. ** P = 0.007 (one way ANOVA). Changes in tumor volumes in female rats treated with AMG386 ( n = 6) or with placebo ( n = 5) for 14 days as determined by anatomical MRI, normalized to the day 0 value. Box plots show 25 th to 75 th percentiles (box) and 5 th to 95 th percentiles (whiskers). The line in the box represents the median. P = 0.0673, not significant (one way ANOVA). ADC values of the rat PitNETs before (day 0) and after (day 14) treatment with AMG386 (red, n = 8) or placebo (blue, n = 5). Box plots show 25 th to 75 th percentiles (box) and 5 th to 95 th percentiles (whiskers). The line in the box represents the median. ** P = 0.009; n.s., not significant ( t ‐test). Ex vivo expression of Tie2, P‐Tie2, Ki67 and Annexin V in PitNETs of female rats treated with AMG386 or placebo for 14 days. Original magnification: 200×; scale bar: 50 µm. Panels shown are representative of the two animal groups. Quantification of Annexin V intensity from tissues stained as in (F) ( n = 4/group). Three different areas per tumor sample were analyzed. Intensities are expressed as arbitrary units ± SEM. *** P

    Techniques Used: Inhibition, In Vivo, Derivative Assay, Magnetic Resonance Imaging, Ex Vivo, Expressing, Staining

    Tumor cells‐secreted Angpt2 induces angiogenesis in vivo Zebrafish larvae were implanted with red fluorescence‐stained GH3 cells infected with unspecific shRNA (shCtrl) or with sh Angpt2 (#2). In the bottom panels, the red channel was omitted to highlight the tumor‐induced microvascular networks. Digital magnifications of graft regions (white boxes) are shown in the bottom left panels. Scale bar: 100 µm. Quantification of tumor‐induced vessel sprouting in zebrafish embryos engrafted with GH3 ‐shCtrl versus shC2 cells ( n = 20 each). Data were normalized against the mean of the control (shCtrl) group arbitrarily set to 1. A.U. arbitrary Units; * P = 0.0319 ( t ‐test). Example of flow cytometry data of primary rat pituitary cells from a tumor‐bearing MENX rat gated for cell surface Tie2 and CD31 expression (cells were pre‐gated for Tie2). Percentage of Tie2 + CD31 − (PitNET cells) and Tie2 + CD31 + (ECs) in the pituitary glands of 7 age‐matched (8 months) MENX rats. Data are expressed as the mean ± SEM. **** P
    Figure Legend Snippet: Tumor cells‐secreted Angpt2 induces angiogenesis in vivo Zebrafish larvae were implanted with red fluorescence‐stained GH3 cells infected with unspecific shRNA (shCtrl) or with sh Angpt2 (#2). In the bottom panels, the red channel was omitted to highlight the tumor‐induced microvascular networks. Digital magnifications of graft regions (white boxes) are shown in the bottom left panels. Scale bar: 100 µm. Quantification of tumor‐induced vessel sprouting in zebrafish embryos engrafted with GH3 ‐shCtrl versus shC2 cells ( n = 20 each). Data were normalized against the mean of the control (shCtrl) group arbitrarily set to 1. A.U. arbitrary Units; * P = 0.0319 ( t ‐test). Example of flow cytometry data of primary rat pituitary cells from a tumor‐bearing MENX rat gated for cell surface Tie2 and CD31 expression (cells were pre‐gated for Tie2). Percentage of Tie2 + CD31 − (PitNET cells) and Tie2 + CD31 + (ECs) in the pituitary glands of 7 age‐matched (8 months) MENX rats. Data are expressed as the mean ± SEM. **** P

    Techniques Used: In Vivo, Fluorescence, Staining, Infection, shRNA, Flow Cytometry, Expressing

    Pharmacological Ang/Tie2 pathway inhibition reduces the viability of PitNET cell lines, and of rat/human primary cultures in vitro Cell proliferation of GH3 cells treated with 5 μg/ml AMG386 (A) or 5 μM Tie2‐KI (B) or left untreated (ctrl) normalized against untreated cells. *** P
    Figure Legend Snippet: Pharmacological Ang/Tie2 pathway inhibition reduces the viability of PitNET cell lines, and of rat/human primary cultures in vitro Cell proliferation of GH3 cells treated with 5 μg/ml AMG386 (A) or 5 μM Tie2‐KI (B) or left untreated (ctrl) normalized against untreated cells. *** P

    Techniques Used: Inhibition, In Vitro

    Knockout of Tie2 in PitNET cells suppresses tumor growth in vivo Scheme of the wild‐type Tie2 receptor (left), and the receptor domain left‐over in the targeted clones #18 and #19 (right) and the impact of the mutation on the activation of downstream signaling. Expression of total Fak and P‐Fak (Y397) in serum‐starved GH3 Ctrl‐KO and Tie2‐KO clones #18 and #19 stimulated with rhANGPT2 for 30 min or left untreated. The numbers represent the ratio phospho/total Fak. Anti‐α‐tubulin antibody was used to check for equal loading. Blot shown is representative of 3 independent experiments. Scheme of the in vivo study in mouse xenografts of GH3 Ctrl‐KO and Tie2‐KO cells (clone #19). T2‐weighted images of two xenografted tumors taken at day 0 and 21 (largest diameter) representing the two animal groups. Scale bar: 2 mm, except GH3‐Ctrl KO day 21: 4 mm. Changes in tumor volumes as determined by MRI were normalized to the day 0 value (=100%) for each animal. All box plots show 25 th to 75 th percentiles (box) and 5 th to 95 th percentiles (whiskers). The line in the box represents the median. ** P = 0.003 (one way ANOVA). Expression of Tie2 (red) in the xenografted tumors ( n = 3 each group). Nuclei were counterstained with DAPI (blue). Original magnification: 400×; scale bar: 50 µm. Source data are available online for this figure.
    Figure Legend Snippet: Knockout of Tie2 in PitNET cells suppresses tumor growth in vivo Scheme of the wild‐type Tie2 receptor (left), and the receptor domain left‐over in the targeted clones #18 and #19 (right) and the impact of the mutation on the activation of downstream signaling. Expression of total Fak and P‐Fak (Y397) in serum‐starved GH3 Ctrl‐KO and Tie2‐KO clones #18 and #19 stimulated with rhANGPT2 for 30 min or left untreated. The numbers represent the ratio phospho/total Fak. Anti‐α‐tubulin antibody was used to check for equal loading. Blot shown is representative of 3 independent experiments. Scheme of the in vivo study in mouse xenografts of GH3 Ctrl‐KO and Tie2‐KO cells (clone #19). T2‐weighted images of two xenografted tumors taken at day 0 and 21 (largest diameter) representing the two animal groups. Scale bar: 2 mm, except GH3‐Ctrl KO day 21: 4 mm. Changes in tumor volumes as determined by MRI were normalized to the day 0 value (=100%) for each animal. All box plots show 25 th to 75 th percentiles (box) and 5 th to 95 th percentiles (whiskers). The line in the box represents the median. ** P = 0.003 (one way ANOVA). Expression of Tie2 (red) in the xenografted tumors ( n = 3 each group). Nuclei were counterstained with DAPI (blue). Original magnification: 400×; scale bar: 50 µm. Source data are available online for this figure.

    Techniques Used: Knock-Out, In Vivo, Clone Assay, Mutagenesis, Activation Assay, Expressing, Magnetic Resonance Imaging

    PitNET cells express Angpt2 and Tie2, and intrinsic Angpt2 promotes PitNET cell survival in vitro Expression of Angpt2 and Tie2 was assessed in Att20, GH3, LβT2 and αT3 cells by western blotting (WB) using specific antibodies. α‐Tubulin was included as loading control. Immunofluorescence (IF) of GH3 cells for Angpt2 (red) and Tie2 (green). Nuclei were counterstained with DAPI (blue). Original magnification: 400x; scale bar: 20 µm. Panels shown are representative of 3 independent experiments. Cell proliferation of GH3 cells transfected with si Angpt2 or scrRNA POOLs and incubated with rhANGPT2 (+rhANGPT2) or left untreated (−rhANGPT2) normalized against scrRNA‐transfected cells. *** P
    Figure Legend Snippet: PitNET cells express Angpt2 and Tie2, and intrinsic Angpt2 promotes PitNET cell survival in vitro Expression of Angpt2 and Tie2 was assessed in Att20, GH3, LβT2 and αT3 cells by western blotting (WB) using specific antibodies. α‐Tubulin was included as loading control. Immunofluorescence (IF) of GH3 cells for Angpt2 (red) and Tie2 (green). Nuclei were counterstained with DAPI (blue). Original magnification: 400x; scale bar: 20 µm. Panels shown are representative of 3 independent experiments. Cell proliferation of GH3 cells transfected with si Angpt2 or scrRNA POOLs and incubated with rhANGPT2 (+rhANGPT2) or left untreated (−rhANGPT2) normalized against scrRNA‐transfected cells. *** P

    Techniques Used: In Vitro, Expressing, Western Blot, Immunofluorescence, Transfection, Incubation

    37) Product Images from "Administration of a Probiotic Mixture Ameliorates Cisplatin-Induced Mucositis and Pica by Regulating 5-HT in Rats"

    Article Title: Administration of a Probiotic Mixture Ameliorates Cisplatin-Induced Mucositis and Pica by Regulating 5-HT in Rats

    Journal: Journal of Immunology Research

    doi: 10.1155/2021/9321196

    Probiotic mixture reduced 5-HT-associated bacteria and inhibited 5-HT secretion by RIN-14B cells. (a) Correlations between the abundances of differential species and colonic 5-HT level. Abundances of Clostridium cluster IV (b) and XIVa (c) in the feces of rats in different groups detected by quantitative PCR. Data are presented as the mean logarithm of bacterial colony-forming units (CFU) per gram of feces. (d) Levels of 5-HT released from RIN-14B cells after exposure to various bacteria or cisplatin. Data are expressed as mean ± SEM ( n = 3–5): ∗ p
    Figure Legend Snippet: Probiotic mixture reduced 5-HT-associated bacteria and inhibited 5-HT secretion by RIN-14B cells. (a) Correlations between the abundances of differential species and colonic 5-HT level. Abundances of Clostridium cluster IV (b) and XIVa (c) in the feces of rats in different groups detected by quantitative PCR. Data are presented as the mean logarithm of bacterial colony-forming units (CFU) per gram of feces. (d) Levels of 5-HT released from RIN-14B cells after exposure to various bacteria or cisplatin. Data are expressed as mean ± SEM ( n = 3–5): ∗ p

    Techniques Used: Real-time Polymerase Chain Reaction

    38) Product Images from "The bromodomain inhibitor JQ1+ reduces calcium-sensing receptor activity in pituitary cell lines"

    Article Title: The bromodomain inhibitor JQ1+ reduces calcium-sensing receptor activity in pituitary cell lines

    Journal: Journal of Molecular Endocrinology

    doi: 10.1530/JME-21-0030

    NPS-2143 has no effect on AtT20 proliferation but does reduce calcium-sensing receptor (CaSR)-mediated cAMP and Ca 2+ i signalling in AtT20 cells. (A) Effect of NPS-2143 (0–1000 nM) on proliferation of AtT20 cells 2 days and 7 days post-treatment. Data were expressed relative to proliferation at day 1. Statistical analyses comparing proliferation between day 2 and day 7 in each group. There was no significant difference between cell proliferation in cells exposed to different concentrations of NPS-2143. Raw fluorescence units ranged between 17,096 and 69,941 for day 2 and 19,935 and 222,377 for day 7. (B) cAMP-response element (CRE) luciferase reporter responses at 5 mM Ca 2+ e in AtT20 cells exposed to DMSO or 20 nM NPS-2143 for 12 h. Data were expressed relative to responses in DMSO-treated cells. (C) Ca 2+ e -induced Fluo-4 intracellular calcium mobilisation assays in AtT20 cells following exposure to DMSO or 20 nM NPS-2143. Raw fluorescence units ranged between 1896 and 58,337. (D) Area under the curve (AUC) of data in C, (E) Maximal Ca 2+ i responses, and (F) EC 50 values showing mean ± 95% CIs obtained in Fluo-4 intracellular calcium mobilisation assays shown in panel C. **** P
    Figure Legend Snippet: NPS-2143 has no effect on AtT20 proliferation but does reduce calcium-sensing receptor (CaSR)-mediated cAMP and Ca 2+ i signalling in AtT20 cells. (A) Effect of NPS-2143 (0–1000 nM) on proliferation of AtT20 cells 2 days and 7 days post-treatment. Data were expressed relative to proliferation at day 1. Statistical analyses comparing proliferation between day 2 and day 7 in each group. There was no significant difference between cell proliferation in cells exposed to different concentrations of NPS-2143. Raw fluorescence units ranged between 17,096 and 69,941 for day 2 and 19,935 and 222,377 for day 7. (B) cAMP-response element (CRE) luciferase reporter responses at 5 mM Ca 2+ e in AtT20 cells exposed to DMSO or 20 nM NPS-2143 for 12 h. Data were expressed relative to responses in DMSO-treated cells. (C) Ca 2+ e -induced Fluo-4 intracellular calcium mobilisation assays in AtT20 cells following exposure to DMSO or 20 nM NPS-2143. Raw fluorescence units ranged between 1896 and 58,337. (D) Area under the curve (AUC) of data in C, (E) Maximal Ca 2+ i responses, and (F) EC 50 values showing mean ± 95% CIs obtained in Fluo-4 intracellular calcium mobilisation assays shown in panel C. **** P

    Techniques Used: Fluorescence, Luciferase

    JQ1+ reduces calcium-sensing receptor (CaSR)-mediated calcium mobilisation in AtT20 cell lines. (A) Ca 2+ e -induced Fluo-4 intracellular calcium mobilisation assays in AtT20 cells following exposure to DMSO, JQ1− and JQ1+ for 24 h. (B) Area under the curve (AUC) of data in A. (C) Maximal Ca 2+ i responses and (D) EC 50 values obtained in Fluo-4 intracellular calcium mobilisation assays shown in panel A. Data are expressed as mean ± s.e.m. in panels A and B, and mean ± 95% CIs in panel C. Statistical analyses compared between groups in panel B, *** P
    Figure Legend Snippet: JQ1+ reduces calcium-sensing receptor (CaSR)-mediated calcium mobilisation in AtT20 cell lines. (A) Ca 2+ e -induced Fluo-4 intracellular calcium mobilisation assays in AtT20 cells following exposure to DMSO, JQ1− and JQ1+ for 24 h. (B) Area under the curve (AUC) of data in A. (C) Maximal Ca 2+ i responses and (D) EC 50 values obtained in Fluo-4 intracellular calcium mobilisation assays shown in panel A. Data are expressed as mean ± s.e.m. in panels A and B, and mean ± 95% CIs in panel C. Statistical analyses compared between groups in panel B, *** P

    Techniques Used:

    (CaSR) is downregulated by JQ1+ treatment. (A) qRT-PCR analysis of CaSR following exposure to cell culture media, DMSO, JQ1− and JQ1+ for 96 h in AtT20 cells. (B) Representative Western blot of CaSR in AtT20 cells following exposure to DMSO, JQ1− and JQ1+ for 96 h. (C) Densitometry analysis of protein expression following treatment with DMSO, JQ1− and JQ1+ for 96 h. CaSR protein expression was expressed relative to calnexin. Data are expressed as mean ± s.e.m. in panels A and C. Statistical analyses compared to DMSO-treated cells in all panels: *** P
    Figure Legend Snippet: (CaSR) is downregulated by JQ1+ treatment. (A) qRT-PCR analysis of CaSR following exposure to cell culture media, DMSO, JQ1− and JQ1+ for 96 h in AtT20 cells. (B) Representative Western blot of CaSR in AtT20 cells following exposure to DMSO, JQ1− and JQ1+ for 96 h. (C) Densitometry analysis of protein expression following treatment with DMSO, JQ1− and JQ1+ for 96 h. CaSR protein expression was expressed relative to calnexin. Data are expressed as mean ± s.e.m. in panels A and C. Statistical analyses compared to DMSO-treated cells in all panels: *** P

    Techniques Used: Quantitative RT-PCR, Cell Culture, Western Blot, Expressing

    JQ1+ reduces cAMP signalling in AtT20 cell lines. (A) Ca 2+ e -induced cAMP-response element (CRE) luciferase reporter responses in AtT20 and HEK-CaSR cell lines. Elevations in Ca 2+ e concentrations increase CRE luciferase in AtT20 cells and reduce luciferase in HEK-CaSR cells. (B) Ca 2+ e -induced CRE luciferase reporter responses in AtT20 cell lines following exposure to DMSO, JQ1− and JQ1+ for 24 h. (C) Maximal CRE luciferase responses from panel B. Data are expressed as mean ± s.e.m. in all panels. Statistical analyses compared to basal responses in each cell line in panel A and to DMSO-treated cells in panel B. Firefly values ranged from 6027 to 981,659 luminescent units and renilla from 1027 to 5031 luminescent units. **** P
    Figure Legend Snippet: JQ1+ reduces cAMP signalling in AtT20 cell lines. (A) Ca 2+ e -induced cAMP-response element (CRE) luciferase reporter responses in AtT20 and HEK-CaSR cell lines. Elevations in Ca 2+ e concentrations increase CRE luciferase in AtT20 cells and reduce luciferase in HEK-CaSR cells. (B) Ca 2+ e -induced CRE luciferase reporter responses in AtT20 cell lines following exposure to DMSO, JQ1− and JQ1+ for 24 h. (C) Maximal CRE luciferase responses from panel B. Data are expressed as mean ± s.e.m. in all panels. Statistical analyses compared to basal responses in each cell line in panel A and to DMSO-treated cells in panel B. Firefly values ranged from 6027 to 981,659 luminescent units and renilla from 1027 to 5031 luminescent units. **** P

    Techniques Used: Luciferase

    JQ1+ does not reduce proliferation of AtT20 cells after 24 h but does decrease calcium-sensing receptor (CaSR) protein expression. (A) Relative proliferation of AtT20 cells following exposure to DMSO, JQ1− and JQ1+ for 24 and 96 h. Data were expressed relative to cell numbers at day 0. (B) Representative Western blot of CaSR in AtT20 cells following exposure to DMSO, JQ1− and JQ1+ for 24 h. (C) Densitometry analysis of protein expression following treatment with DMSO, JQ1− and JQ1+ for 24 h. CaSR protein expression was expressed relative to calnexin. Data are expressed as mean ± s.e.m. in panels A and C. Statistical analyses compared to DMSO treatment in panel A, *** P
    Figure Legend Snippet: JQ1+ does not reduce proliferation of AtT20 cells after 24 h but does decrease calcium-sensing receptor (CaSR) protein expression. (A) Relative proliferation of AtT20 cells following exposure to DMSO, JQ1− and JQ1+ for 24 and 96 h. Data were expressed relative to cell numbers at day 0. (B) Representative Western blot of CaSR in AtT20 cells following exposure to DMSO, JQ1− and JQ1+ for 24 h. (C) Densitometry analysis of protein expression following treatment with DMSO, JQ1− and JQ1+ for 24 h. CaSR protein expression was expressed relative to calnexin. Data are expressed as mean ± s.e.m. in panels A and C. Statistical analyses compared to DMSO treatment in panel A, *** P

    Techniques Used: Expressing, Western Blot

    39) Product Images from "Drp1-Mediated Mitochondrial Metabolic Dysfunction Inhibits the Tumor Growth of Pituitary Adenomas"

    Article Title: Drp1-Mediated Mitochondrial Metabolic Dysfunction Inhibits the Tumor Growth of Pituitary Adenomas

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2022/5652586

    Drp1 expression was increased in human LI-PA samples, and overexpressed Drp1 affected the proliferation and apoptosis of PA cell lines. (a) Expression of Drp1 mRNA was assessed by RT-qPCR in human pituitary gland RNA standard, low proliferation index PA (LI-PA) tissues ( n = 10), and high proliferation index PA (HI-PA) tissues ( n = 10). (b) Expression of the Drp1 protein was assessed by IHC staining (left) in LI-PA ( n = 10) and HI-PA ( n = 10) sections. Statistical analysis of the IHC staining (right). Scale bar, 50 μ m. (c) Drp1 mRNA and protein levels were measured in the control (Vector) group and Drp1-overexpressing (Drp1-OE) group GH3 cells by RT-qPCR and western blotting. (d) Vector and Drp1-OE GH3 cells were incubated with/without 10 μ M Mdivi-1 for 0, 24, 48, or 72 h, and cell proliferation was assessed at different time points by a CCK-8 assay ( n = 6, ± SEM) in each group. (e) Wild-type (WT) MMQ cells were incubated with/without Mdivi-1 (10 μ M) for 0, 24, 48, or 72 h. Cell proliferation of the two groups at different time points was assessed by a CCK-8 assay ( n = 6, ± SEM). (f) The treatment to MMQ cells was carried out with wild-type AtT-20 cells. (g) Vector and Drp1-OE GH3 cells were infected with NC shRNA or Drp1 shRNA (Drp1 shRNA1 or Drp1 shRNA3). Then, cell pr oliferation was assessed at 0, 24, 48, or 72 h by a CCK-8 assay in each group ( n = 6, ± SEM). (h) Vector and Drp1-OE GH3 cells were incubated with/without 10 μ M Mdivi-1 for 48 h. Cell apoptosis was measured by flow cytometry in each group ( n = 3, ± SEM). (i) Vector and Drp1-OE GH3 cells were infected with NC shRNA or Drp1 shRNA (Drp1 shRNA1 or Drp1 shRNA3). Cell apoptosis was measured by flow cytometry in each group ( n = 3, ± SEM). (j) The colocalization of Drp1 and mitochondria in GH3 cells by using immunofluorescence staining. Representative immunostaining of Drp1 (Green) and TOMM20 (Red) was observed by laser confocal microscopy in the vector and Drp1-OE groups. Yellow points represent overlay of Drp1 and mitochondria. Scale bar, 7.5 μ m. (k) Protein expression level of Drp1 in isolated mitochondria was detected by western blotting in the vector and Drp1-OE groups. An unpaired t -test was used to assess statistical significance. ∗ P
    Figure Legend Snippet: Drp1 expression was increased in human LI-PA samples, and overexpressed Drp1 affected the proliferation and apoptosis of PA cell lines. (a) Expression of Drp1 mRNA was assessed by RT-qPCR in human pituitary gland RNA standard, low proliferation index PA (LI-PA) tissues ( n = 10), and high proliferation index PA (HI-PA) tissues ( n = 10). (b) Expression of the Drp1 protein was assessed by IHC staining (left) in LI-PA ( n = 10) and HI-PA ( n = 10) sections. Statistical analysis of the IHC staining (right). Scale bar, 50 μ m. (c) Drp1 mRNA and protein levels were measured in the control (Vector) group and Drp1-overexpressing (Drp1-OE) group GH3 cells by RT-qPCR and western blotting. (d) Vector and Drp1-OE GH3 cells were incubated with/without 10 μ M Mdivi-1 for 0, 24, 48, or 72 h, and cell proliferation was assessed at different time points by a CCK-8 assay ( n = 6, ± SEM) in each group. (e) Wild-type (WT) MMQ cells were incubated with/without Mdivi-1 (10 μ M) for 0, 24, 48, or 72 h. Cell proliferation of the two groups at different time points was assessed by a CCK-8 assay ( n = 6, ± SEM). (f) The treatment to MMQ cells was carried out with wild-type AtT-20 cells. (g) Vector and Drp1-OE GH3 cells were infected with NC shRNA or Drp1 shRNA (Drp1 shRNA1 or Drp1 shRNA3). Then, cell pr oliferation was assessed at 0, 24, 48, or 72 h by a CCK-8 assay in each group ( n = 6, ± SEM). (h) Vector and Drp1-OE GH3 cells were incubated with/without 10 μ M Mdivi-1 for 48 h. Cell apoptosis was measured by flow cytometry in each group ( n = 3, ± SEM). (i) Vector and Drp1-OE GH3 cells were infected with NC shRNA or Drp1 shRNA (Drp1 shRNA1 or Drp1 shRNA3). Cell apoptosis was measured by flow cytometry in each group ( n = 3, ± SEM). (j) The colocalization of Drp1 and mitochondria in GH3 cells by using immunofluorescence staining. Representative immunostaining of Drp1 (Green) and TOMM20 (Red) was observed by laser confocal microscopy in the vector and Drp1-OE groups. Yellow points represent overlay of Drp1 and mitochondria. Scale bar, 7.5 μ m. (k) Protein expression level of Drp1 in isolated mitochondria was detected by western blotting in the vector and Drp1-OE groups. An unpaired t -test was used to assess statistical significance. ∗ P

    Techniques Used: Expressing, Quantitative RT-PCR, Immunohistochemistry, Staining, Plasmid Preparation, Western Blot, Incubation, CCK-8 Assay, Infection, shRNA, Flow Cytometry, Immunofluorescence, Immunostaining, Confocal Microscopy, Isolation

    40) Product Images from "Increased expression and retention of the secretory chaperone proSAAS following cell stress"

    Article Title: Increased expression and retention of the secretory chaperone proSAAS following cell stress

    Journal: Cell Stress & Chaperones

    doi: 10.1007/s12192-020-01128-7

    ER stress induced by thapsigargin increases cellular BiP and proSAAS levels while suppressing proSAAS secretion. AtT-20 cells ( a ), Neuro2A cells ( b ), and primary cortical cells ( c ) were exposed to thapsigargin (Thap) for 24 h, and cellular lysates and the conditioned medium were assessed for proSAAS expression (hippocampal medium proSAAS was below the limit of detection and is thus not shown). Immunoblots probed with BiP and proSAAS antiserum were normalized to β-actin as a loading control. Data are presented as mean ± SD of four replicates. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; one-way ANOVA and with the Tukey post hoc test. i.r., immunoreactivity
    Figure Legend Snippet: ER stress induced by thapsigargin increases cellular BiP and proSAAS levels while suppressing proSAAS secretion. AtT-20 cells ( a ), Neuro2A cells ( b ), and primary cortical cells ( c ) were exposed to thapsigargin (Thap) for 24 h, and cellular lysates and the conditioned medium were assessed for proSAAS expression (hippocampal medium proSAAS was below the limit of detection and is thus not shown). Immunoblots probed with BiP and proSAAS antiserum were normalized to β-actin as a loading control. Data are presented as mean ± SD of four replicates. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; one-way ANOVA and with the Tukey post hoc test. i.r., immunoreactivity

    Techniques Used: Expressing, Western Blot

    ER stress induced by tunicamycin increases the expression of BiP and proSAAS mRNAs. Tunicamycin (Tun) exposure increases mRNA levels of the ER stress marker BiP in AtT-20 cells ( a ), Neuro2A cells ( b ), and primary hippocampal neurons ( c ) but increases proSAAS mRNA levels only in Neuro2A cells . Cells were treated with increasing concentrations of tunicamycin (Tun) for 24 h, and mRNA levels were determined by quantitative real-time PCR. Each mRNA is shown relative to GAPDH and plotted as the fold increase compared with the vehicle control (DMSO). The data shown represent the averages of four biological and three technical replicates ± SD. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; one-way ANOVA with the Tukey post hoc test
    Figure Legend Snippet: ER stress induced by tunicamycin increases the expression of BiP and proSAAS mRNAs. Tunicamycin (Tun) exposure increases mRNA levels of the ER stress marker BiP in AtT-20 cells ( a ), Neuro2A cells ( b ), and primary hippocampal neurons ( c ) but increases proSAAS mRNA levels only in Neuro2A cells . Cells were treated with increasing concentrations of tunicamycin (Tun) for 24 h, and mRNA levels were determined by quantitative real-time PCR. Each mRNA is shown relative to GAPDH and plotted as the fold increase compared with the vehicle control (DMSO). The data shown represent the averages of four biological and three technical replicates ± SD. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; one-way ANOVA with the Tukey post hoc test

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

    ER stress induced by tunicamycin increases cellular BiP and proSAAS protein while suppressing proSAAS secretion. Immunoblot of cell lysate and conditioned media collected from AtT-20 cells ( a ), Neuro2A cells ( b ), and primary hippocampal neurons ( c ) treated with increasing concentrations of tunicamycin (Tun) for 24 h. Immunoblots probed with BiP and proSAAS antiserum were normalized to β-actin as a loading control. Data are presented as mean ± SD of three replicates. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; one-way ANOVA with the Tukey post hoc test. i.r., immunoreactivity
    Figure Legend Snippet: ER stress induced by tunicamycin increases cellular BiP and proSAAS protein while suppressing proSAAS secretion. Immunoblot of cell lysate and conditioned media collected from AtT-20 cells ( a ), Neuro2A cells ( b ), and primary hippocampal neurons ( c ) treated with increasing concentrations of tunicamycin (Tun) for 24 h. Immunoblots probed with BiP and proSAAS antiserum were normalized to β-actin as a loading control. Data are presented as mean ± SD of three replicates. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; one-way ANOVA with the Tukey post hoc test. i.r., immunoreactivity

    Techniques Used: Western Blot

    ER stress induced by thapsigargin increases the expression of BiP and proSAAS mRNAs. The effect of the ER calcium stressor thapsigargin (Thap) on mRNA levels of BIP and proSAAS in AtT-20 cells ( a ) and Neuro2A ( b ) cells and primary cortical cells ( c ). Cells were treated with increasing concentrations of thapsigargin for 24 h, and mRNA levels were determined by quantitative real-time PCR. Each mRNA is shown relative to GAPDH and plotted as the fold increase compared with the vehicle control (DMSO). The data shown represent the averages of four biological and three technical replicates ± SD. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; one-way ANOVA with the Tukey post hoc test
    Figure Legend Snippet: ER stress induced by thapsigargin increases the expression of BiP and proSAAS mRNAs. The effect of the ER calcium stressor thapsigargin (Thap) on mRNA levels of BIP and proSAAS in AtT-20 cells ( a ) and Neuro2A ( b ) cells and primary cortical cells ( c ). Cells were treated with increasing concentrations of thapsigargin for 24 h, and mRNA levels were determined by quantitative real-time PCR. Each mRNA is shown relative to GAPDH and plotted as the fold increase compared with the vehicle control (DMSO). The data shown represent the averages of four biological and three technical replicates ± SD. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; one-way ANOVA with the Tukey post hoc test

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    ProSAAS overexpression decreases tunicamycin sensitivity, while proSAAS knockout increases sensitivity. a Overexpression of proSAAS in three AtT-20 cell clones was verified by immunoblotting; results were normalized to actin. b Wild-type and proSAAS-overexpressing clones were treated with 0.5 μg/ml of tunicamycin (Tun) or vehicle for 24 h and cell viability assessed. c Loss of proSAAS expression in AtT-20-KO cell clones is shown by PCR following reverse transcription of mRNA as the loss of about 600 bp proSAAS PCR product. d Wild-type and knockout clones were treated with 0.25 μg/ml and 0.5 μg/ml of tunicamycin (Tun) or vehicle for 24 h and cell viability assessed. WST-1 results are expressed as percentages of corresponding non-Tun-treated control cells . Data are presented as mean ± SD of six replicates. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; two-way ANOVA with the Tukey post hoc test
    Figure Legend Snippet: ProSAAS overexpression decreases tunicamycin sensitivity, while proSAAS knockout increases sensitivity. a Overexpression of proSAAS in three AtT-20 cell clones was verified by immunoblotting; results were normalized to actin. b Wild-type and proSAAS-overexpressing clones were treated with 0.5 μg/ml of tunicamycin (Tun) or vehicle for 24 h and cell viability assessed. c Loss of proSAAS expression in AtT-20-KO cell clones is shown by PCR following reverse transcription of mRNA as the loss of about 600 bp proSAAS PCR product. d Wild-type and knockout clones were treated with 0.25 μg/ml and 0.5 μg/ml of tunicamycin (Tun) or vehicle for 24 h and cell viability assessed. WST-1 results are expressed as percentages of corresponding non-Tun-treated control cells . Data are presented as mean ± SD of six replicates. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; two-way ANOVA with the Tukey post hoc test

    Techniques Used: Over Expression, Knock-Out, Clone Assay, Expressing, Polymerase Chain Reaction

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    ATCC att 20 cell line
    Cell-specific chromatin landscapes determine cell-selective GR occupancy (a–b) Pituitary-specific GR occupancy dictated by pituitary-specific DNaseI sensitivity transitions. Shown are examples of DNaseI sensitivity and GR occupancy patterns in relation to hormone exposure comparing mouse mammary (3134) and pituitary <t>(AtT-20)</t> cells (see Fig.1 legend and Supplementary Fig.8a-c for additional examples). (c) Global GR occupancy vs. chromatin accessibility landscape in pituitary cells. In pituitary cells, virtually all sites of GR occupancy (94.9%, 3,079/3,242 sites) occur within pre-hormone accessible chromatin. The small fraction of re-progra mmed GR sites (138 GR ChIP peaks, 4.2% of total) is shown in red. As in mammary cells, only a small fraction of pre-hormone accessible chromatin is occupied (note: for legibility, GR circle shown at 5X scale). (d) Significant differences in genomic distribution of pre-hormone DNaseI sensitivity in mammary (grey) vs. pituitary (green) cells; only 0.78% of genome (20.5Mb) is accessible in both cell types. (e) GR occupancy is highly cell-selective. Only 371 GR occupancy sites are shared between mammary and pituitary cells (4.5% of 3134 sites and 11.4% of AtT-20 sites).
    Att 20 Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell-specific chromatin landscapes determine cell-selective GR occupancy (a–b) Pituitary-specific GR occupancy dictated by pituitary-specific DNaseI sensitivity transitions. Shown are examples of DNaseI sensitivity and GR occupancy patterns in relation to hormone exposure comparing mouse mammary (3134) and pituitary (AtT-20) cells (see Fig.1 legend and Supplementary Fig.8a-c for additional examples). (c) Global GR occupancy vs. chromatin accessibility landscape in pituitary cells. In pituitary cells, virtually all sites of GR occupancy (94.9%, 3,079/3,242 sites) occur within pre-hormone accessible chromatin. The small fraction of re-progra mmed GR sites (138 GR ChIP peaks, 4.2% of total) is shown in red. As in mammary cells, only a small fraction of pre-hormone accessible chromatin is occupied (note: for legibility, GR circle shown at 5X scale). (d) Significant differences in genomic distribution of pre-hormone DNaseI sensitivity in mammary (grey) vs. pituitary (green) cells; only 0.78% of genome (20.5Mb) is accessible in both cell types. (e) GR occupancy is highly cell-selective. Only 371 GR occupancy sites are shared between mammary and pituitary cells (4.5% of 3134 sites and 11.4% of AtT-20 sites).

    Journal: Nature genetics

    Article Title: Chromatin accessibility pre-determines glucocorticoid receptor binding patterns

    doi: 10.1038/ng.759

    Figure Lengend Snippet: Cell-specific chromatin landscapes determine cell-selective GR occupancy (a–b) Pituitary-specific GR occupancy dictated by pituitary-specific DNaseI sensitivity transitions. Shown are examples of DNaseI sensitivity and GR occupancy patterns in relation to hormone exposure comparing mouse mammary (3134) and pituitary (AtT-20) cells (see Fig.1 legend and Supplementary Fig.8a-c for additional examples). (c) Global GR occupancy vs. chromatin accessibility landscape in pituitary cells. In pituitary cells, virtually all sites of GR occupancy (94.9%, 3,079/3,242 sites) occur within pre-hormone accessible chromatin. The small fraction of re-progra mmed GR sites (138 GR ChIP peaks, 4.2% of total) is shown in red. As in mammary cells, only a small fraction of pre-hormone accessible chromatin is occupied (note: for legibility, GR circle shown at 5X scale). (d) Significant differences in genomic distribution of pre-hormone DNaseI sensitivity in mammary (grey) vs. pituitary (green) cells; only 0.78% of genome (20.5Mb) is accessible in both cell types. (e) GR occupancy is highly cell-selective. Only 371 GR occupancy sites are shared between mammary and pituitary cells (4.5% of 3134 sites and 11.4% of AtT-20 sites).

    Article Snippet: The AtT-20 cell line is an anterior pituitary corticotroph of murine origin (ATCC).

    Techniques: Chromatin Immunoprecipitation

    Regulatory motifs in GR-occupied regions differ substantially between cell types (a–b) Results of de novo motif discovery (see Supplementary Notes ) performed on the top 500 GR occupancy sites identified in 3134 (panel a ) and AtT-20 (panel b ). The GR sites were further separated into pre-programmed (GR occupancy within pre-hormone accessible chromatin) vs. re-programmed (GR occupancy within pre-hormone inaccessible chromatin) sites. Shown are motifs with highly significant enrichment (e

    Journal: Nature genetics

    Article Title: Chromatin accessibility pre-determines glucocorticoid receptor binding patterns

    doi: 10.1038/ng.759

    Figure Lengend Snippet: Regulatory motifs in GR-occupied regions differ substantially between cell types (a–b) Results of de novo motif discovery (see Supplementary Notes ) performed on the top 500 GR occupancy sites identified in 3134 (panel a ) and AtT-20 (panel b ). The GR sites were further separated into pre-programmed (GR occupancy within pre-hormone accessible chromatin) vs. re-programmed (GR occupancy within pre-hormone inaccessible chromatin) sites. Shown are motifs with highly significant enrichment (e

    Article Snippet: The AtT-20 cell line is an anterior pituitary corticotroph of murine origin (ATCC).

    Techniques:

    Sites of calcium transients during mitosis . Calcium transients correspond with sites of CaV1 immunofluorescence in AtT-20 Cells during mitosis. Simultaneous imaging of (a) Fluo4 (4 μ M) intracellular calcium transients and (b) cell morphology by DIC in AtT-20 cells during cytokinesis. Regions: 1=midbody; 2, 3=spindle mid-zone; 4=nuclear region of interphase cell; 5=background (media only). (c) Plot of fluorescence units for each region over time (sec). Regions of enhanced calcium transients correspond to midbody (1) and spindle mid-zone (2, 3). Break in X-axis marks time of acquisition following loading of Fluo-4.

    Journal: International Journal of Cell Biology

    Article Title: Cell Cycle-Dependent Localization of Voltage-Dependent Calcium Channels and the Mitotic Apparatus in a Neuroendocrine Cell Line(AtT-20)

    doi: 10.1155/2009/487959

    Figure Lengend Snippet: Sites of calcium transients during mitosis . Calcium transients correspond with sites of CaV1 immunofluorescence in AtT-20 Cells during mitosis. Simultaneous imaging of (a) Fluo4 (4 μ M) intracellular calcium transients and (b) cell morphology by DIC in AtT-20 cells during cytokinesis. Regions: 1=midbody; 2, 3=spindle mid-zone; 4=nuclear region of interphase cell; 5=background (media only). (c) Plot of fluorescence units for each region over time (sec). Regions of enhanced calcium transients correspond to midbody (1) and spindle mid-zone (2, 3). Break in X-axis marks time of acquisition following loading of Fluo-4.

    Article Snippet: AtT-20 cells are cultured in Dulbecco's Modified Eagle medium (DMEM) containing 4.5 gm glucose/L and 10 percent fetal bovine serum.

    Techniques: Immunofluorescence, Imaging, Fluorescence

    Calcium channel CaV1.2 and CaV1.3 localization in mitotic cells . 1(A) compares CaV1.2 staining in AtT-20 cells (a)–(c), PC-12 cells (d), and INS-1 cells (e)–(g). CaV1.2 is located with kinetechores at the “poleward side” during metaphase and anaphase ((a), (b): inset) in the AtT-20 cells. Midbody CaV1.2 staining is observed in both AtT-20 cells and PC-12 cells (c), (d) but not in INS-1 cells (g). 1(B) is a similar comparison for CaV1.3 and shows staining at the mid-spindle zone only in AtT-20s during telophase (a). In contrast, all three neuroendocrine cell types display CaV1.3 staining at the midbody during cytokinesis ((B): (b), (c), and (e)). 1(C) shows control staining of Cos 7 cells; in particular, note that there is no nonspecific staining at the midbody of these cells (1(C) (c), (e)). CaV1.2 and CaV1.3 are stained with antibodies specific to alpha subunit sequences for CaV1.2 and CaV1.3 (see Methods, Alamone Labs) and detected with secondary goat anti-rabbit antibody Alexa 555 (red); Microtubules are stained with DM1A and goat anti-mouse Alexa 647 (blue); kinetechores are stained with anti-CREST antibody and Alexa 488 (green). Bleedthrough between microtubule and CREST staining channels was corrected for as described in methods.

    Journal: International Journal of Cell Biology

    Article Title: Cell Cycle-Dependent Localization of Voltage-Dependent Calcium Channels and the Mitotic Apparatus in a Neuroendocrine Cell Line(AtT-20)

    doi: 10.1155/2009/487959

    Figure Lengend Snippet: Calcium channel CaV1.2 and CaV1.3 localization in mitotic cells . 1(A) compares CaV1.2 staining in AtT-20 cells (a)–(c), PC-12 cells (d), and INS-1 cells (e)–(g). CaV1.2 is located with kinetechores at the “poleward side” during metaphase and anaphase ((a), (b): inset) in the AtT-20 cells. Midbody CaV1.2 staining is observed in both AtT-20 cells and PC-12 cells (c), (d) but not in INS-1 cells (g). 1(B) is a similar comparison for CaV1.3 and shows staining at the mid-spindle zone only in AtT-20s during telophase (a). In contrast, all three neuroendocrine cell types display CaV1.3 staining at the midbody during cytokinesis ((B): (b), (c), and (e)). 1(C) shows control staining of Cos 7 cells; in particular, note that there is no nonspecific staining at the midbody of these cells (1(C) (c), (e)). CaV1.2 and CaV1.3 are stained with antibodies specific to alpha subunit sequences for CaV1.2 and CaV1.3 (see Methods, Alamone Labs) and detected with secondary goat anti-rabbit antibody Alexa 555 (red); Microtubules are stained with DM1A and goat anti-mouse Alexa 647 (blue); kinetechores are stained with anti-CREST antibody and Alexa 488 (green). Bleedthrough between microtubule and CREST staining channels was corrected for as described in methods.

    Article Snippet: AtT-20 cells are cultured in Dulbecco's Modified Eagle medium (DMEM) containing 4.5 gm glucose/L and 10 percent fetal bovine serum.

    Techniques: Staining

    Nifedipine attenuates AtT-20 Cell density . (a) Cell viability . Incubation with the DHP CaV antagonist, nifedipine, results in a dose-dependent decrease in AtT-20 cell number using the colorimetric MTS assay for cell viability. The half maximal dose for this effect is 1.5 ± 0.3 μ M nifedipine. Graph shows normalized data from 3 experiments. (b) Cell proliferation and mitotic stages. Graph shows mean number of cells ±SE for at least 5 fields (≥ 800 total cells counted for Day 3 for each condition). There is a significant decrease in the total number of cells counted between vehicle control and nifedipine-treated cells (* P

    Journal: International Journal of Cell Biology

    Article Title: Cell Cycle-Dependent Localization of Voltage-Dependent Calcium Channels and the Mitotic Apparatus in a Neuroendocrine Cell Line(AtT-20)

    doi: 10.1155/2009/487959

    Figure Lengend Snippet: Nifedipine attenuates AtT-20 Cell density . (a) Cell viability . Incubation with the DHP CaV antagonist, nifedipine, results in a dose-dependent decrease in AtT-20 cell number using the colorimetric MTS assay for cell viability. The half maximal dose for this effect is 1.5 ± 0.3 μ M nifedipine. Graph shows normalized data from 3 experiments. (b) Cell proliferation and mitotic stages. Graph shows mean number of cells ±SE for at least 5 fields (≥ 800 total cells counted for Day 3 for each condition). There is a significant decrease in the total number of cells counted between vehicle control and nifedipine-treated cells (* P

    Article Snippet: AtT-20 cells are cultured in Dulbecco's Modified Eagle medium (DMEM) containing 4.5 gm glucose/L and 10 percent fetal bovine serum.

    Techniques: Incubation, MTS Assay

    Effect of actinomycin D on dexamethasone regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 4).

    Journal: The Journal of Neuroscience

    Article Title: Region-Specific Regulation of RGS4 (Regulator of G-Protein–Signaling Protein Type 4) in Brain by Stress and Glucocorticoids: In Vivo and In VitroStudies

    doi: 10.1523/JNEUROSCI.19-10-03674.1999

    Figure Lengend Snippet: Effect of actinomycin D on dexamethasone regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 4).

    Article Snippet: AtT20 cells were derived from mouse pituitary and exhibit many properties of pituitary corticotrophs (ACTH-secreting cells).

    Techniques: Expressing

    Time course study for dexamethasone regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 3–6; * p

    Journal: The Journal of Neuroscience

    Article Title: Region-Specific Regulation of RGS4 (Regulator of G-Protein–Signaling Protein Type 4) in Brain by Stress and Glucocorticoids: In Vivo and In VitroStudies

    doi: 10.1523/JNEUROSCI.19-10-03674.1999

    Figure Lengend Snippet: Time course study for dexamethasone regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 3–6; * p

    Article Snippet: AtT20 cells were derived from mouse pituitary and exhibit many properties of pituitary corticotrophs (ACTH-secreting cells).

    Techniques: Expressing

    Dose–response analyses for dexamethasone ( Dex ) regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 3–6; * p

    Journal: The Journal of Neuroscience

    Article Title: Region-Specific Regulation of RGS4 (Regulator of G-Protein–Signaling Protein Type 4) in Brain by Stress and Glucocorticoids: In Vivo and In VitroStudies

    doi: 10.1523/JNEUROSCI.19-10-03674.1999

    Figure Lengend Snippet: Dose–response analyses for dexamethasone ( Dex ) regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells. Data are expressed as the mean percent of control (± SEM; n = 3–6; * p

    Article Snippet: AtT20 cells were derived from mouse pituitary and exhibit many properties of pituitary corticotrophs (ACTH-secreting cells).

    Techniques: Expressing

    Comparison of the regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells by dexamethasone ( Dex ), CRF, and forskolin ( FK ). Data are expressed as the mean percent of control (± SEM; n = 4; * p

    Journal: The Journal of Neuroscience

    Article Title: Region-Specific Regulation of RGS4 (Regulator of G-Protein–Signaling Protein Type 4) in Brain by Stress and Glucocorticoids: In Vivo and In VitroStudies

    doi: 10.1523/JNEUROSCI.19-10-03674.1999

    Figure Lengend Snippet: Comparison of the regulation of RGS4 mRNA expression in CATH.a ( A ) and AtT20 ( B ) cells by dexamethasone ( Dex ), CRF, and forskolin ( FK ). Data are expressed as the mean percent of control (± SEM; n = 4; * p

    Article Snippet: AtT20 cells were derived from mouse pituitary and exhibit many properties of pituitary corticotrophs (ACTH-secreting cells).

    Techniques: Expressing