Journal: Molecular and Cellular Biology

Article Title: Transcription of the mouse secretory protease inhibitor p12 gene is activated by the developmentally regulated positive transcription factor Sp1.

doi: 10.1128/mcb.12.9.3796

Figure Lengend Snippet: FIG. 5. Transient transfection analyses. A plasmid (p12.108) in which the minimal p12 promoter region from positions +7 to -108 was inserted immediately upstream from the CAT reporter gene and a mutated version of the same construct (pl2.108/M), containing point mutations for the nucleotide positions found critical for binding of the trans-acting proteins to p12.A, were transiently transfected into pituitary GH4C1 cells, human HepG2, rat Hepa7.6, and mouse CL-2 hepatoma cells, monkey kidney COS-1 cells, and human HeLa cells. Levels of CAT activity were measured and normalized to the amount of hGH secreted as described in Materials and Methods. Values are relative to the level of CAT activity directed by the control plasmid p12.108 (100%) and are shown along with the standard deviation measured for each transfected plasmid in all cell lines tested. CAT activities directed by plasmid pRSVCAT are also included as positive controls.

Article Snippet: Rat pituitary GH4C1 (kindly provided by David D. Moore, Massachusetts General Hospital, Boston), AtT-20 (ATCC CCL89), and human hepatoma HepG2 (ATCC HB 8065) cells, a variant of rat hepatoma Morris 7777 cells (Hepa7.6; kindly provided by Luc Belanger, Centre de Recherche de l'Hotel Dieu de Quebec, Quebec, Canada) (18), and mouse liver BNL CL.2 (ATCC TIB 73), monkey kidney COS-1 (ATCC CRL 1650) and CV1 (ATCC CCL70), rat fibroblast Rat2 (ATCC CRL1764), human prostate DU145 (ATCC HTB81), rat Sertoli TM4 (ATCC CRL1715), and human epitheloid carcinoma HeLa (ATCC CCL 2) cells were all grown in Dulbecco's modified Eagle's medium (GIBCO) supplemented with 10% fetal bovine serum.

Techniques: Transfection, Plasmid Preparation, Construct, Binding Assay, Activity Assay, Control, Standard Deviation

Journal: Molecular and Cellular Biology

Article Title: Transcription of the mouse secretory protease inhibitor p12 gene is activated by the developmentally regulated positive transcription factor Sp1.

doi: 10.1128/mcb.12.9.3796

Figure Lengend Snippet: FIG. 7. p12.A-binding activity in nuclear extracts from tissue culture cell lines versus normal mouse tissues. (A) Crude nuclear extracts were prepared from HeLa, AtT-20, GH4C1, CV1, Rat2, CL-2, Hepa7.6, DU145, and TM4 cells as described previously (56). Proteins (5 p,g) from each nuclear extract were incubated with the 5'-end-labeled p12.A-containing probe (2 x 104 cpm) described in the legend to Fig. 1 under identical conditions. Formation of the DNA-protein complexes was analyzed by electrophoresis through 6% nondenaturing polyacrylamide gels in Tris-glycine buffer. The position of each shifted complex (Rl to R4) is indicated along with the position of the unbound fraction of the probe (U). (B) Same as panel A except that crude nuclear extracts were prepared from whole mouse tissues (testis, lung, kidney, pancreas, liver, brain, thymus, and prostate). The position of each shifted complex (Rl to R4) is indicated along with the positions of the unbound fraction of the probe (U) and a set of FMCs. Lane P, labeled probe alone.

Article Snippet: Rat pituitary GH4C1 (kindly provided by David D. Moore, Massachusetts General Hospital, Boston), AtT-20 (ATCC CCL89), and human hepatoma HepG2 (ATCC HB 8065) cells, a variant of rat hepatoma Morris 7777 cells (Hepa7.6; kindly provided by Luc Belanger, Centre de Recherche de l'Hotel Dieu de Quebec, Quebec, Canada) (18), and mouse liver BNL CL.2 (ATCC TIB 73), monkey kidney COS-1 (ATCC CRL 1650) and CV1 (ATCC CCL70), rat fibroblast Rat2 (ATCC CRL1764), human prostate DU145 (ATCC HTB81), rat Sertoli TM4 (ATCC CRL1715), and human epitheloid carcinoma HeLa (ATCC CCL 2) cells were all grown in Dulbecco's modified Eagle's medium (GIBCO) supplemented with 10% fetal bovine serum.

Techniques: Binding Assay, Activity Assay, Incubation, Labeling, Electrophoresis

Journal: OncoTargets and Therapy

Article Title: Resveratrol decreases B-cell lymphoma-2 expression and viability in GH3 pituitary adenoma cells of the rat

doi: 10.2147/ott.s45154

Figure Lengend Snippet: Figure 3 Ethidium bromide-stained agarose gel (1%) showing electrophoretic mobility of 10 µg genomic DNA, which had been freshly isolated from GH3 cells treated with 100 µM resveratrol (R) for 48 hours, or with the corresponding concentration of 0.1% of the solvent ethanol (E) or solely with cell-culture medium (M). λ-DNA digested with the restriction endonuclease Hind-III served as a molecular weight standard ranging from 2 to 23.1 kb. It can be seen that the DNA isolated from the medium or solvent controls migrates as a bright spot of high-molecular-weight genomic DNA of approximately 20 kb, while this spot disappeared in resveratrol- treated cells and was replaced by a more continuous faint staining along the whole lane, thus indicating severe DNA degradation. The continuous staining of this lane was clearly visible under the ultraviolet illumination. It cannot be seen ideally on the photograph, due to the large differences in fluorescence/cm2 compared to the high- molecular-weight DNA in lanes E and M. Four independent observers confirmed this homogeneous stain, but did not detect any laddering. This result indicated that most of the cells had died by a nonapoptotic mode of cell death.

Article Snippet: This is well known to the scientific community, and MTT assays can thus even be exploited to measure cell adherence.11 We found that GH3 cells, in accordance with the supplier’s (ATCC) description, were loosely adherent.

Techniques: Staining, Agarose Gel Electrophoresis, Isolation, Concentration Assay, Solvent, Cell Culture, Molecular Weight, High Molecular Weight, Fluorescence

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Brusatol Inhibits Tumor Growth and Increases the Efficacy of Cabergoline against Pituitary Adenomas

doi: 10.1155/2021/6696015

Figure Lengend Snippet: Brusatol (BT) repressed the growth and hormone secretion of pituitary adenomas. (a, b) GH3 and MMQ cells were treated with a range of concentrations of BT for 24 and 48 h. Cell viability was determined by MTS. (c, d) GH3 and MMQ cells were treated with BT for 24 h; formed colonies were photographed and counted using the ImageJ software. (e) Different subtypes of primary pituitary tumor cells were treated with 0.25 μ M BT for 24 h; cell viability was determined by MTS. (f) After treating GH3 and MMQ cells with BT (0–500 nM) for 24 h, culture medium supernatants were collected for ELISA to determine the prolactin hormone concentration. (g) After treating GH3 cells with BT (0–500 nM) for 24 h, culture medium supernatants were collected for ELISA to determine the levels of GH hormone. Data are represented as the mean ± SD. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

Article Snippet: Rat pituitary tumor GH3 cell lines (ATCC CRL-10609) and MMQ cell lines (ATCC CRL-10609) were purchased from the American Type Culture Collection (ATCC).

Techniques: Software, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Brusatol Inhibits Tumor Growth and Increases the Efficacy of Cabergoline against Pituitary Adenomas

doi: 10.1155/2021/6696015

Figure Lengend Snippet: Brusatol (BT) inhibits pituitary tumor growth in xenograft models. (a, d) Representative images of xenograft tumors from mice treated with control vehicle or BT at 24 days of drug administration. (b, e) Tumor volume growth curves of nude mice in different treatment groups. BT inhibited the rate of tumor growth in GH3 and MMQ cells. (c, f) Tumor weights from mice injected with GH3 cells and treated with control vehicle or BT at day 24 of drug administration. Data are represented as the mean ± SD. ∗ P < 0.05.

Article Snippet: Rat pituitary tumor GH3 cell lines (ATCC CRL-10609) and MMQ cell lines (ATCC CRL-10609) were purchased from the American Type Culture Collection (ATCC).

Techniques: Control, Injection

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Brusatol Inhibits Tumor Growth and Increases the Efficacy of Cabergoline against Pituitary Adenomas

doi: 10.1155/2021/6696015

Figure Lengend Snippet: Brusatol- (BT-) induced apoptotic cell death in GH3 and MMQ cells. (a, b) Induction of apoptosis in GH3 and MMQ cells with BT treatment (250 nM) for 24 h followed by Annexin V and PI staining. Apoptosis ratios were measured by flow cytometry. (c, d) After 0–24 h treatment with BT (250 nM) in GH3 and MMQ cells, Western blot analysis was used to monitor the expression level of c-caspase-3, c-caspase-8, and Bcl-2. (e, f) GH3 and MMQ cells were treated with BT or NAC (100 μ M) alone or in combination for 24 h and MTS assays conducted to determine cell viability. Data are represented as the mean ± SD. ∗ P < 0.05.

Article Snippet: Rat pituitary tumor GH3 cell lines (ATCC CRL-10609) and MMQ cell lines (ATCC CRL-10609) were purchased from the American Type Culture Collection (ATCC).

Techniques: Staining, Flow Cytometry, Western Blot, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Brusatol Inhibits Tumor Growth and Increases the Efficacy of Cabergoline against Pituitary Adenomas

doi: 10.1155/2021/6696015

Figure Lengend Snippet: Brusatol (BT) downregulated the phosphorylation level of 4EBP1 and S6K1 in pituitary adenomas. (a) After 0–24 h treatment with BT (250 nM) in GH3 and MMQ cells, Western blot analysis was used to monitor the expression level of 4EBP1, S6K1, p-4EBP1, and p-S6K1. (b) Primary pituitary tumor cells (1 GHoma and 1 nonfunctional tumor) were treated with BT; Western blot analysis revealed the expression level of 4EBP1, S6K1, p-4EBP1, and p-S6K1. (c) Representative images of IHC-stained samples show that BT decreased p-4EBP1 and p-S6k1 expression in GH3 and MMQ xenograft models; scale bar, 100 μ m.

Article Snippet: Rat pituitary tumor GH3 cell lines (ATCC CRL-10609) and MMQ cell lines (ATCC CRL-10609) were purchased from the American Type Culture Collection (ATCC).

Techniques: Phospho-proteomics, Western Blot, Expressing, Staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Brusatol Inhibits Tumor Growth and Increases the Efficacy of Cabergoline against Pituitary Adenomas

doi: 10.1155/2021/6696015

Figure Lengend Snippet: Brusatol (BT) increased ROS accumulation in GH3 and MMQ cells by inhibiting the Nrf2 pathway. (a) GH3 and MMQ cells treated with BT (250 nM) for 24 h were analyzed by flow cytometry after DCFH-DA staining to determine ROS levels. (b) GH3 and MMQ cells were treated with a range of concentrations of BT for 24 h. Western blot analysis showed that BT downregulated the expression of Nrf2 and its downstream gene Ho-1 but had no effect on Keap1. (c) GH3 and MMQ cell lines were treated with BT (250 nM) and/or NAC (100 μ M) for 24 h and cell viability measured by MTS assay. (d) GH3 and MMQ cell lines were treated with BT (250 nM) and/or NAC (100 μ M) for 24 h; Western blot analysis showed that NAC reversed the inhibitory effect of BT on inhibition of p-4EBP1 and p-S6K1. Data are presented as the mean ± SD. ∗∗ P < 0.01; ∗∗∗ P < 0.001.

Article Snippet: Rat pituitary tumor GH3 cell lines (ATCC CRL-10609) and MMQ cell lines (ATCC CRL-10609) were purchased from the American Type Culture Collection (ATCC).

Techniques: Flow Cytometry, Staining, Western Blot, Expressing, MTS Assay, Inhibition

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Brusatol Inhibits Tumor Growth and Increases the Efficacy of Cabergoline against Pituitary Adenomas

doi: 10.1155/2021/6696015

Figure Lengend Snippet: Brusatol (BT) increased the cytotoxicity of cabergoline in GH3 and MMQ cells. (a) GH3 and MMQ cells were treated with BT (250 nM) and/or cabergoline (50 μ M) for 24 h. Cell viability was measured by MTS assays, and (c) Western blot analysis was conducted to determine the level of p-4EBP1 and p-S6K1. (b) Representative images of colony formation assays. GH3 and MMQ cells were treated with BT (250 nM) and/or cabergoline (50 μ M) for 24 h. Colony formation was allowed to proceed for 14 days. Data are presented as the mean ± SD. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

Article Snippet: Rat pituitary tumor GH3 cell lines (ATCC CRL-10609) and MMQ cell lines (ATCC CRL-10609) were purchased from the American Type Culture Collection (ATCC).

Techniques: Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Brusatol Inhibits Tumor Growth and Increases the Efficacy of Cabergoline against Pituitary Adenomas

doi: 10.1155/2021/6696015

Figure Lengend Snippet: Brusatol (BT) augmented the cytotoxic effect of cabergoline in GH3 xenografts. (a) The 4 groups were treated with BT or/and cabergoline every other day. After 30 days of treatment, the nude mice were anesthetized, and the tumors were harvested. (b) Tumor volume was measured every 3 days (volume = π /6 × [larger diameter] × [smaller diameter] 2 ), and the volume curve was determined using GraphPad Prism 8.0. (c) Comparison of the average xenograft tumor weight. Data are represented as the mean ± SD. ∗ P < 0.05; ∗∗∗ P < 0.001.

Article Snippet: Rat pituitary tumor GH3 cell lines (ATCC CRL-10609) and MMQ cell lines (ATCC CRL-10609) were purchased from the American Type Culture Collection (ATCC).

Techniques: Comparison

Journal: Diabetes research and clinical practice

Article Title: Functional analysis of PCSK2 coding variants: a founder effect in the Old Order Amish population

doi: 10.1016/j.diabres.2017.06.023

Figure Lengend Snippet: Panel A) Diagram of the human PC2 protein indicating the positions of the PCSK2 coding variants studied here (grey ovals) and other known coding variants (yellow rectangles). Panel B) In vitro activity assay in CHO-7B2 cells (a constitutively-secreting cell line). Conditioned medium obtained from transfected CHO-7B2 cells was subjected to in vitro enzymatic assays. Panel C) Expression and secretion of PCSK2 variants in CHO-7B2 cells. Immunoblot analysis of proPC2 and 7B2 protein levels in whole cell lysates and in the overnight conditioned medium obtained from transfected CHO-7B2 cells. Ponceau S staining was used as a loading control. Note the comparable levels of activity for all variants except A267T; this variant was largely retained within the cell. Panel D) Cleavage of proglucagon by wild-type and variant enzymes in GH4C1 cells (a neuroendocrine cell line). Glucagon levels were measured by RIA in the overnight conditioned medium of GH4C1 cells co-transfected with proglucagon and PCSK2 variant cDNAs; an immunoblot of PC2 protein levels in the same conditioned medium samples is shown above. Note the lack of A267T PC2 in the medium, indicative of impaired secretion; glucagon production by other variants was similar to that of wild-type PC2. Enzyme activity and RIA data were analyzed using a one-way ANOVA followed by a post hoc Bonferroni’s test (***, P < 0.001); n = 3. Panel E) The R430W variant enzyme exhibits a broadened pH curve. The activity of wild-type PC2 and R430W-containing PC2 was measured following dilution into buffers designed to reach the pHs shown. The values have been normalized to the maximum activity at pH 5.0 to correct for differences in expression across different experiments. Note the increased activity of the R430W PC2 mutant at higher pHs. Enzyme activity was analyzed using a two-way ANOVA followed by a post-hoc Bonferroni’s test (***, P<0.001; mean ± SD for 4 independent experiments).

Article Snippet: Rat pituitary tumor GH4C1 cells were obtained from ATCC and were maintained in high glucose DMEM, supplemented with 10% FBS, in humidified 5% CO 2 at 37°C.

Techniques: In Vitro, Activity Assay, Transfection, Expressing, Western Blot, Staining, Control, Variant Assay, Mutagenesis