Journal: Cancers

Article Title: A Novel ZIP4-HDAC4-VEGFA Axis in High-Grade Serous Ovarian Cancer

doi: 10.3390/cancers13153821

Figure Lengend Snippet: ZIP4 sensitized HDACi in HGSOC cells. ( A ). Established PEA2-ZIP4-KD cell lines. Other ZIP4-KO/KD, NOTCH3-KO cell lines used in this work were established and published previously [ , ]. The up- or downregulation of ZIP4 in these cell lines, including T80-ZIP4-OE, were confirmed and shown in A. ( B – D ). ZIP4-OE in T80 (an immortalized human ovarian surface epithelial cell line) cells sensitized to TSA (0.3 and 1 µM), PANO (1 and 3 µM), and LMK-235 (3–10 µM). ZIP4-KO/KD in both PE04 and PEA2 cells resulted in more resistance to HDACis-induced cell death. ( E ). FACS-sorted ZIP4 + cells respond to LMK-235 (3 and 10 μM) significantly stronger than those ZIP4 − cells. The MTT values for each individual cell line with solvent treatment were used as 100% in all results shown in this figure. All experiments were repeated ≥ three times and presented as means ± standard deviation (SD). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: VALPROIC ACID (Catalog No: 2815100) was from Fisher Scientific Company LLC (Hanover Park, IL, USA); Entinostat (Catalog No: A8171-50) and Pracinostat (Catalog No: A4095-5) were from APEXBIO TECHNOLOGY LLC (HOUSTON, TX, USA); Mocetinostat (HY-12164) was from MEDCHEMEXPRESS LLC (Monmouth Junction, NJ, USA); TSA (Catalog No: S1045), LMK-235 (Catalog No: S7569), Belinostat (Catalog No:S1085), and panobinostat (PANO, LBH589; Catalog No: S1030) were from Selleck Chemicals LLC (Munich, Germany).

Techniques: Solvent, Standard Deviation

Journal: Cancers

Article Title: A Novel ZIP4-HDAC4-VEGFA Axis in High-Grade Serous Ovarian Cancer

doi: 10.3390/cancers13153821

Figure Lengend Snippet: HDAC4-KD inhibited HDACi sensitization in PE04 and PEA2 cells. ( A ). HDAC4-KD PE04 and PEA2 cell lines generated using shRNAs. ( B ). HDAC4-KD PE04 and PEA2 cell lines reduced cell numbers (measured by MTT). ( C – E ). HDAC4-KD increased cell resistance to HDACis (TSA, PANO and LMK-235). The MTT values for each individual cell line with solvent treatment were used as 100% in ( B – D ). All experiments were repeated ≥ three times and are presented as means ± standard deviation (SD). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: VALPROIC ACID (Catalog No: 2815100) was from Fisher Scientific Company LLC (Hanover Park, IL, USA); Entinostat (Catalog No: A8171-50) and Pracinostat (Catalog No: A4095-5) were from APEXBIO TECHNOLOGY LLC (HOUSTON, TX, USA); Mocetinostat (HY-12164) was from MEDCHEMEXPRESS LLC (Monmouth Junction, NJ, USA); TSA (Catalog No: S1045), LMK-235 (Catalog No: S7569), Belinostat (Catalog No:S1085), and panobinostat (PANO, LBH589; Catalog No: S1030) were from Selleck Chemicals LLC (Munich, Germany).

Techniques: Generated, Solvent, Standard Deviation

Journal: Cancers

Article Title: A Novel ZIP4-HDAC4-VEGFA Axis in High-Grade Serous Ovarian Cancer

doi: 10.3390/cancers13153821

Figure Lengend Snippet: HDACis inhibited spheroid formation and HDAC4 gene compensated the spheroid formation activity in ZIP4-KO/KD cells. ( A , B ). HDACis (TSA, PANO, and LMK-235) inhibited spheroid formation in PE04 and PEA2 cells. The scale bars = 400 nm. Summary of quantified spheroid formation data from > three independent experiments in PE04 and PEA2 cells. ( C ) . HDAC4 gene transfected cells increased cell numbers (measured by MTT), compared to vector-transfected ZIP4-KO/KD cells. ( D , E ). HDAC4 gene transfected green cells compensated for the spheroid formation activity lost in ZIP4-KO/KD PE04 and PEA2 cells. The scale bars = 1000 nm. For quantification, cell aggregates < 70 nm and/or with irregular shapes were not counted as spheroids. Representative pictures are shown. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: VALPROIC ACID (Catalog No: 2815100) was from Fisher Scientific Company LLC (Hanover Park, IL, USA); Entinostat (Catalog No: A8171-50) and Pracinostat (Catalog No: A4095-5) were from APEXBIO TECHNOLOGY LLC (HOUSTON, TX, USA); Mocetinostat (HY-12164) was from MEDCHEMEXPRESS LLC (Monmouth Junction, NJ, USA); TSA (Catalog No: S1045), LMK-235 (Catalog No: S7569), Belinostat (Catalog No:S1085), and panobinostat (PANO, LBH589; Catalog No: S1030) were from Selleck Chemicals LLC (Munich, Germany).

Techniques: Activity Assay, Transfection, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Caloric restriction and resveratrol promote longevity through the Sirtuin-1-dependent induction of autophagy

doi: 10.1038/cddis.2009.8

Figure Lengend Snippet: Role of Sirtuin-1 (Sirt-1) in starvation-induced autophagy. ( a , b ) Sirt-1 requirement for the induction of autophagy by nutrient deprivation in cancer cells. Wild-type (WT) HCT 116 cells were transfected with a GFP-LC3-encoding construct for 24 h and then cultured for additional 6 h in nutrient-free (NF) conditions or treated with 1 μ M rapamycin (Rapa), 2.5 μ M tunicamycin (Tun), 30 μ M cyclic pifithrin-α (Pif-α) in the presence or absence of 100 μM EX527 ( a ). ( b ) Alternatively, WT HCT 116 cells were transfected with control or a Sirt-1-depleting siRNA and subsequently with a plasmid for the expression of GFP-LC3 for 24 h, followed by culture in autophagy-inducing conditions as in a . The percentage of cells exhibiting the accumulation of GFP-LC3 in cytoplasmic puncta (GFP-LC3 vac ) is reported (mean±S.E.M., n =3, * P <0.05). ( c ) Requirement of the C. elegans Sirtuin-1 ortholog, SIR-2.1, for starvation-induced autophagy. Autophagy was assayed in WT animals and sir-2.1(ok434) deletion mutants expressing the DsRed::LGG-1 transgene, which were fed ad libitum (AL), grown under conditions of dietary restriction (DR), or treated with 2 μ g/ml Tun or 1 μg/ml Rapa. Columns illustrate mean pixel intensity of DsRed::LGG1 (mean±S.E.M., n =3, * P <0.05). ( d ) WT and p53 −/− HCT 116 cells were transfected with the indicated siRNAs, re-transfected with a GFP-LC3-encoding plasmid for 24 h and then cultured for further 6 h in control or starvation conditions. Columns depict the percentage of GFP-LC3 vac cells (mean±S.E.M., n =3, * P <0.05). ( e ) Immunoprecipitation of endogenous p53 and lysine-acetylated proteins. HCT 116 cells of the indicated genotype were cultured in starvation conditions or treated with 70 nM trichostatin (TSA, positive control for acetylation) for 6 h, followed by immunoprecipitation of p53 or lysine-acetylated proteins and immunoblotting for the detection of p53 acetylated on Lys382 (Ac Lys382 p53), total p53, or ATG7. Alternatively, total proteins were assayed for ATG7 abundance and LC3 maturation. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels were determined to monitor the equal loading of lanes. Results are representative of three independent experiments. Numbers next to bands indicate MW (kDa)

Article Snippet: Cells were seeded in 6- or 12-well plates or in 10 cm dishes and grown for 24 h before treatment with 1 μ M Rapa, 100 μ M EX527, and 70 nM trichostatin (all from Tocris Bioscience, Ellisville, MO, USA); 30 μ M cyclin pifithrin-α (Calbiochem, Nottingham, UK); and 100 μ M Resv, 2.5 μ M Tun, 1 nM BafA1, and 100 nM leupeptin (all from Sigma-Aldrich), which lasted 6 h unless otherwise stated.

Techniques: Transfection, Construct, Cell Culture, Control, Plasmid Preparation, Expressing, Immunoprecipitation, Positive Control, Western Blot

Journal: BMC Molecular Biology

Article Title: Signal transducer and activator of transcription STAT5 is recruited to c - Myc super-enhancer

doi: 10.1186/s12867-016-0063-y

Figure Lengend Snippet: c - Myc gene expression in Ba/F3-derived cell lines. a STAT5 protein and phosphorylation levels in the parental Ba/F3 cells and in Ba/F3 cells stably expressing STAT5A-WT (Ba/F3-WT) and STAT5A-1*6 (Ba/F3-1*6). Ba/F3 and Ba/F3-WT cells, which grow in IL-3-containing medium, were withdrawn from IL-3 for 11 h and stimulated with IL-3 for 15 min. IL-3-independent Ba/F3-1*6 cells were stimulated with IL-3 in parallel. Brij whole-cell protein lysates (20 μg) were analysed by Western blot using antibodies specific for phosphorylated STAT5 (pSTAT5), total STAT5A and STAT5B (STAT5A/B), transgenic STAT5A-WT and STAT5A-1*6 (FLAG), and α-tubulin as a loading control. STAT5A/B signal in parental Ba/F3 cells corresponds to endogenous STAT5 protein levels, while the signals detected in the stable cell lines Ba/F3-WT and Ba/F3-1*6 represent both endogenous (STAT5A and STAT5B) and transgenic (STAT5A-WT or -1*6) proteins. b STAT5-mediated expression of c - Myc in Ba/F3-WT and -1*6 cells. Rested Ba/F3-WT cells stably expressing wild-type STAT5A were pre-treated 30 min with 200 nM TSA and further stimulated with IL-3 for 30 min. Ba/F3-1*6 cells expressing constitutively active STAT5A-1*6 were treated in parallel with 200 nM TSA for 60 min. Expression of STAT5 target genes ( Cis , Osm , c - Myc ) and of the housekeeping gene 36b4 was analysed by RT-qPCR. Expression of c - Myc , like that of Cis and Osm , was induced by STAT5A-WT and by constitutively active STAT5A-1*6, in an IL-3-dependent and -independent manner respectively. STAT5A-WT- and STAT5A-1*6-mediated expression of c - Myc , Cis and Osm was inhibited by the deacetylase inhibitor trichostatin A (TSA). Student’s t tests were employed to compare on the one hand IL-3-induced (WT) or 1*6-induced gene expression to the unstimulated WT control, and on the other hand TSA-treated to the vehicle control in each condition; ** P < 0.01, *** P < 0.001, **** P < 0.0001; a P value <0.05 was considered statistically significant. c Expression of STAT5 target genes is inhibited by both deacetylase (TSA) and BET (JQ1) inhibitors. Ba/F3 cells were pre-treated with 20 nM TSA or 500 nM JQ1 for 30 min and stimulated with IL-3 for 60 min. Expression of STAT5 target genes ( Cis , c - Myc ) and of control genes (IL-3-dependent MAPK target gene JunB and housekeeping gene 36b4 ) was analysed by RT-qPCR, as above. IL-3-induced expression of Cis and c - Myc , but not that of JunB , was inhibited by TSA and JQ1. One-way ANOVA with Dunnett’s multiple comparison test was used to assess differences between TSA- or JQ1-treated conditions and the vehicle-treated IL-3-stimulated control; *** P < 0.001; a P < 0.05 was considered statistically significant

Article Snippet: Dimethyl sulfoxide (DMSO) and trichostatin A (TSA) were purchased from SIGMA (D-2650 and T-8552 respectively). (+)-JQ1 (BPS Bioscience #27401)—hereafter abbreviated to JQ1—was purchased from BIOMOL GmbH.

Techniques: Expressing, Derivative Assay, Stable Transfection, Western Blot, Transgenic Assay, Quantitative RT-PCR, Histone Deacetylase Assay