1,25(OH)₂D₃ induces p27 in MMECs.

<p>(A) qPCR indicated induction of Cdkn1b mRNA at 48 hrs of 100 nM 1,25(OH)₂D₃ (1,25D3, * = p < 0.05) and a non-significant trend toward induction of Cdkn1a at 48 hrs. (B) Induction of p27 protein was detected at 6 and 24 hrs of treatment with 100 nM 1,25(OH)₂D₃ (D) compared to EtOH control (E). The p21 antibody signal was weak.</p

1,25(OH)₂D₃ inhibits MMEC growth.

<p>(A) MMECs were treated with the indicated doses of 1,25(OH)₂D₃ or 0.1% vehicle control (EtOH) for 48 hours. Viable cells were counted according to trypan blue exclusion. * = p < 0.05. (B) Representative images from clonogenic assays, quantified in (C). * = p < 0.05.</p

A Role for Interleukin-1 Alpha in the 1,25 Dihydroxyvitamin D₃ Response in Mammary Epithelial Cells

<div><p>Breast cancer is the most common non-cutaneous malignancy in American women, and better preventative strategies are needed. Epidemiological and laboratory studies point to vitamin D₃ as a promising chemopreventative agent for breast cancer. Vitamin D₃ metabolites induce anti-proliferative effects in breast cancer cells <i>in vitro</i> and <i>in vivo</i>, but few studies have investigated their effects in normal mammary epithelial cells. We hypothesized that 1,25(OH)₂D₃, the metabolically active form of vitamin D₃, is growth suppressive in normal mouse mammary epithelial cells. In addition, we have previously established a role for the cytokine interleukin-1 alpha (IL1α) in the anti-proliferative effects of 1,25(OH)₂D₃ in normal prostate cells, and so we hypothesized that IL1α is involved in the 1,25(OH)₂D₃ response in mammary cells. Evaluation of cell viability, clonogenicity, senescence, and induction of cell cycle regulators p21 and p27 supported an anti-proliferative role for 1,25(OH)₂D₃ in mammary epithelial cells. Furthermore, 1,25(OH)₂D₃ increased the intracellular expression of IL1α, which was necessary for the anti-proliferative effects of 1,25(OH)₂D₃ in mammary cells. Together, these findings support the chemopreventative potential of vitamin D₃ in the mammary gland and present a role for IL1α in regulation of mammary cell proliferation by 1,25(OH)₂D₃.</p> </div

Cellular localization of IL1α and IL1R1 in MMECs.

<p>(A) Punctate IL1α signals (arrows) were detected in the nuclear and cytoplasmic compartments of MMECs upon 24 and 48 hr treatments with 100 nM 1,25(OH)₂D₃. IL1α was undetected in 0.1% vehicle control-treated cells (EtOH) and under negative control conditions (no primary antibody). (B) IL1RI signal was detected at the edges of the cell membranes at 24 and 48 hours in MMECs treated with 0.1% vehicle control (EtOH, arrows). IL1RI was detected both at the edges of the cells and within the cytoplasmic compartments after treatment with 100 nM 1,25(OH)₂D₃ for 48 hrs (arrows). No signal was detected under negative control conditions.</p

1,25(OH)₂D₃ induces IL1α mRNA and protein in MMECs.

<p>(A) qPCR revealed a 5 to 6-fold induction of IL1α mRNA by 100 nM 1,25(OH)₂D₃. * = p < 0.001. (B) 100 nM 1,25(OH)₂D₃ (D) induced IL1α protein at 6, 24, and 48 hours. Very little IL1α was present in the cells treated with ethanol control (E).</p

IL1α mediates the anti-proliferative effects of 1,25(OH)₂D₃ in MMECs.

<p>(A) Western blot for IL1α expression in MMEC clones (cl.) infected with IL1α shRNA or negative control shRNA (shRNA NC). E = 0.1% ethanol, D = 100 nM 1,25(OH)₂D₃. (B) 48 hr trypan blue exclusion assays in MMEC clones infected with IL1α shRNA or control shRNA. * = p < 0.05.</p

1,25(OH)₂D₃ induces senescence of MMECs.

<p>(A) Representative images from the SA-β-gal assays quantified in (B). 100 nM 1,25(OH)₂D₃ significantly induced senescence compared to the control treatment (EtOH). Bars labeled “a” or “b” are statistically significantly different from each other according to ANOVA and post-hoc Fisher’s LSD test (n = 3 replicates, ~160 cells quantified in each of 10 fields of view per replicate, critical value = 0.05). </p

Few-Layer PdSe₂ Sheets: Promising Thermoelectric Materials Driven by High Valley Convergence

Herein, we report a comprehensive study on the structural and electronic properties of bulk, monolayer, and multilayer PdSe₂ sheets. First, we present a benchmark study on the structural properties of bulk PdSe₂ by using 13 commonly used density functional theory (DFT) functionals. Unexpectedly, the most commonly used van der Waals (vdW)-correction methods, including DFT-D2, optB88, and vdW-DF2, fail to provide accurate predictions of lattice parameters compared to experimental data (relative error > 15%). On the other hand, the PBE-TS series functionals provide significantly improved prediction with a relative error of <2%. Unlike hexagonal two-dimensional materials like graphene, transition metal dichalcogenides, and h-BN, the conduction band minimum of monolayer PdSe₂ is not located along the high symmetry lines in the first Brillouin zone; this highlights the importance of the structure–property relationship in the pentagonal lattice. Interestingly, high valley convergence is found in the conduction and valence bands in monolayer, bilayer, and trilayer PdSe₂ sheets, suggesting promising application in thermoelectric cooling

Reaction conditions for measurement of DPP-2, DPP-8, DPP-9, PEP, and FAPα activities.

<p>Reaction conditions for measurement of DPP-2, DPP-8, DPP-9, PEP, and FAPα activities.</p

Biased, Non-equivalent Gene-Proximal and -Distal Binding Motifs of Orphan Nuclear Receptor TR4 in Primary Human Erythroid Cells

<div><p>We previously reported that TR2 and TR4 orphan nuclear receptors bind to direct repeat (DR) elements in the ε- and γ-globin promoters, and act as molecular anchors for the recruitment of epigenetic corepressors of the multifaceted DRED complex, thereby leading to ε- and γ-globin transcriptional repression during definitive erythropoiesis. Other than the ε- and γ-globin and the <i>GATA1</i> genes, TR4-regulated target genes in human erythroid cells remain unknown. Here, we identified TR4 binding sites genome-wide using chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq) as human primary CD34⁺ hematopoietic progenitors differentiated progressively to late erythroid precursors. We also performed whole transcriptome analyses by RNA-seq to identify TR4 downstream targets after lentiviral-mediated TR4 shRNA knockdown in erythroid cells. Analyses from combined ChIP-seq and RNA-seq datasets indicate that DR1 motifs are more prevalent in the proximal promoters of TR4 direct target genes, which are involved in basic biological functions (e.g., mRNA processing, ribosomal assembly, RNA splicing and primary metabolic processes). In contrast, other non-DR1 repeat motifs (DR4, ER6 and IR1) are more prevalent at gene-distal TR4 binding sites. Of these, approximately 50% are also marked with epigenetic chromatin signatures (such as P300, H3K27ac, H3K4me1 and H3K27me3) associated with enhancer function. Thus, we hypothesize that TR4 regulates gene transcription <i>via</i> gene-proximal DR1 sites as TR4/TR2 heterodimers, while it can associate with novel nuclear receptor partners (such as RXR) to bind to distant non-DR1 consensus sites. In summary, this study reveals that the TR4 regulatory network is far more complex than previously appreciated and that TR4 regulates basic, essential biological processes during the terminal differentiation of human erythroid cells.</p></div