Journal: The FASEB Journal

Article Title: Plasma membrane cholesterol trafficking in steroidogenesis

doi: 10.1096/fj.201800697rrr

Figure Lengend Snippet: Figure 1. Plasma membranes can support steroidogenesis. PMs from rat adrenals, MLTCs, Y1 adrenocortical cells, and liver were purified and added to the in vitro mitochondrial reconstitution assay, in the absence of any other sources of cholesterol or accessory proteins. Pregnenolone production was measured by ELISA. A) The relative pregnenolone production was measured with rat adrenal PMs, MLTC, Y1 adrenocortical cell, and liver PMs as the sources of cholesterol. The basal pregnenolone production for mitochondria without PMs ranged from 0.5 to 3 ng/ml. B) Comparison of rat adrenal PMs and lipid emulsion as cholesterol sources for pregnenolone production in the absence or presence of accessory proteins. LE, lipid emulsion; Mito, mitochondria; NS, not significant; SNAREs/StAR, a cocktail containing recombinant SNAP23, SNAP25, a-SNAP, STX17, and N62-StAR. Results are representative of 3 independent experiments (n = 3). **P , 0.01, ***P , 0.001.

Article Snippet: Mouse Leydig tumor cells (MLTCs) were obtained from ATCC and were cultured in RPMI 1640 plus 10% fetal bovine serum, 100 U/ml penicillin, and 100 g/ml streptomycin.

Techniques: Clinical Proteomics, In Vitro, Reconstitution Assay, Enzyme-linked Immunosorbent Assay, Comparison, Emulsion, Recombinant

Journal: eLife

Article Title: KRAB-zinc finger protein gene expansion in response to active retrotransposons in the murine lineage

doi: 10.7554/eLife.56337

Figure Lengend Snippet: ( A ) Differential analysis of summative (all individual insertions combined) H3K9me3 enrichment at TE groups in Chr10-cl, Chr13.1-cl and Chr13.2-cl KO ES cells. TE groups targeted by one or several KRAB-ZFPs encoded within the deleted clusters are highlighted in orange (differential enrichment over the entire TE sequences) and red (differential enrichment at TE regions that overlap with KRAB-ZFP ChIP-seq peaks). ( B ) Top: Schematic view of the Cd59a / Cd59b locus with a 5’ truncated ETn insertion. ChIP-seq (Input subtracted from ChIP) data for overexpressed epitope-tagged Gm13051 (a Chr4-cl KRAB-ZFP) in F9 EC cells, and re-mapped KAP1 (GEO accession: GSM1406445) and H3K9me3 (GEO accession: GSM1327148) in WT ES cells are shown together with RNA-seq data from Chr4-cl WT and KO ES cells (mapped using Bowtie (-a -m 1 -- strata -v 2) to exclude reads that cannot be uniquely mapped). Bottom: Transcriptional activity of a 5 kb fragment with or without fragments of the ETn insertion was tested by luciferase reporter assay in Chr4-cl WT and KO ES cells.

Article Snippet: Cell line ( Mus musculus ) , F9 Embryonic carcinoma cells , ATCC , ATCC CRL-1720 , .

Techniques: ChIP-sequencing, RNA Sequencing, Activity Assay, Luciferase, Reporter Assay

Journal: eLife

Article Title: KRAB-zinc finger protein gene expansion in response to active retrotransposons in the murine lineage

doi: 10.7554/eLife.56337

Figure Lengend Snippet: ( A ) Differential gene expression in Chr2-cl and Chr4-cl KO ES cells. Significantly up- and downregulated genes ( adjusted p-value <0.05) are highlighted in red and green, respectively, KRAB-ZFP genes within the deleted clusters are shown in blue. ( B ) Correlation of TEs and gene deregulation. Plots show enrichment of TE groups within 100 kb of up- and downregulated genes relative to all genes. Significantly overrepresented LTR and LINE groups ( adjusted p-value <0.1) are highlighted in blue and red, respectively. ( C ) Schematic view of the downstream region of Chst1 where a 5’ truncated ETn insertion is located. ChIP-seq (Input subtracted from ChIP) data for overexpressed epitope-tagged Gm13051 (a Chr4-cl KRAB-ZFP) in F9 EC cells, and re-mapped KAP1 (GEO accession: GSM1406445) and H3K9me3 (GEO accession: GSM1327148) in WT ES cells are shown together with RNA-seq data from Chr4-cl WT and KO ES cells (mapped using Bowtie (-a -m 1 -- strata -v 2) to exclude reads that cannot be uniquely mapped). ( D ) RT-qPCR analysis of Chst1 mRNA expression in Chr4-cl WT and KO ES cells with or without the CRISPR/Cas9 deleted ETn insertion near Chst1 . Values represent mean expression (normalized to Gapdh) from three biological replicates per sample (each performed in three technical replicates) in arbitrary units. Error bars represent standard deviation and asterisks indicate significance (p<0.01, Student’s t-test). n.s.: not significant. ( E ) Mean coverage of ChIP-seq data (Input subtracted from ChIP) in Chr4-cl WT and KO ES cells over 127 full-length ETn insertions. The binding sites of the Chr4-cl KRAB-ZFPs Rex2 and Gm13051 are indicated by dashed lines.

Article Snippet: Cell line ( Mus musculus ) , F9 Embryonic carcinoma cells , ATCC , ATCC CRL-1720 , .

Techniques: Gene Expression, ChIP-sequencing, RNA Sequencing, Quantitative RT-PCR, Expressing, CRISPR, Standard Deviation, Binding Assay

Journal: eLife

Article Title: KRAB-zinc finger protein gene expansion in response to active retrotransposons in the murine lineage

doi: 10.7554/eLife.56337

Figure Lengend Snippet:

Article Snippet: Cell line ( Mus musculus ) , F9 Embryonic carcinoma cells , ATCC , ATCC CRL-1720 , .

Techniques: Recombinant, Expressing, Plasmid Preparation, CRISPR, Construct, Sequencing, Software

Journal: Journal of the Endocrine Society

Article Title: Somatic PRKAR1A Gene Mutation in a Nonsyndromic Metastatic Large Cell Calcifying Sertoli Cell Tumor

doi: 10.1210/js.2019-00022

Figure Lengend Snippet: Staining and immunohistochemistry images of the (a-c) resected primary tumor and (d-f) peripheral lymph node with metastasis. (a, b) Sections show a testicular neoplasm composed of nests and cords of large tumor cells with eosinophilic cytoplasm and prominent nucleoli in a background of fibrotic stroma. Mild cell atypia was present, but mitoses or necrosis were not readily visible. (c) The proliferative index as per Ki-67 staining was ∼5%. (d, e) Tissue from metastatic LCCSCT had morphology similar to that of the primary lesion and was positive for inhibin alpha staining (f). Asterisk, normal testis; arrow, LCCSCT; Magnification, 4× (a, d, c), 10× (b), and 40× (e, f).

Article Snippet: The monoclonal mouse anti-human Ki-67 (clone MIB1) antibody (1:200 dilution; Agilent, Santa Clara, CA) [ ] and mouse anti-human inhibin alpha (clone R1) antibody (1:100 dilution; Bio-Rad, Hercules, CA) [ ] were used for immunohistochemistry.

Techniques: Staining, Immunohistochemistry