Journal: eLife

Article Title: Concentration dependent chromatin states induced by the bicoid morphogen gradient

doi: 10.7554/eLife.28275

Figure Lengend Snippet: ( A ) Quanitification of Knirps intensity in wild-type, triple mutant ( hb nos tsl ) and quadruple mutant ( bcd hb nos tsl ). Bicoid activates patterned expression of Knirps. In embryos in which Bicoid is the only source of maternal patterning information ( hb nos tsl ), a broad domain of Kni is expressed in the posterior of the embryo. In quadruple mutant embryos, a low level of uniform Knirps is expressed ubiquitously, suggesting that Bcd is required for activating expression of knirps above a background level. Heat-fixed embryos from wild-type (Oregon-R) mothers, hunchback nanos torso-like germline clones and bicoid hunchback nanos torso-like germline clones were pooled and immunostained in a single tube with a rat anti-Knirps primary antibody and Alexa-647 rat antibody. Embryos were mounted on a single slide and imaged by confocal microscopy. Representative embryos for each genotype are shown. Fluorescence intensity of Knirps was extracted from dorsal profiles of midsagittal sections of embryos and plotted using MATLAB. Data are fluorescence intensity minus background, and error bars are standard error of the mean for n = 5 wild-type, n = 8 hb nos tsl, and n = 6 bcd hb nos tsl embryos. ( B ) Smear plot generated in EdgeR showing the log transformed fold-change in Bcd binding between mutant and wild-type embryos for each Bcd peak, vs. the average log transformed sequencing read counts per million (CPM). Bcd binding shows no significant changes between wild-type and nos tsl mutant embryos. Significance was determined using EdgeR to perform a pairwise exact test with a cutoff of FDR ≤ 0.05, comparing binding between eGFP-Bcd;;bcd E1 and eGFP-Bcd;; bcd E1 hb FB nos L7 tsl 4 in the 1,027 Bcd peaks. ( C ) Schematic of the uniform Bcd transgene. The uniform Bcd transgene contains an N-terminal GFP-tagged Bcd driven by the various maternal promoters discussed in the text. Downstream of the bcd coding sequence is a cassette containing the endogenous bcd 3'UTR and a 3xP3-hsp70 promoter driving promoter of RFP. This cassette is flanked by FRT sites. The sqh 3'UTR lies downstream of the FRT cassette. Flies expressing this version of the transgene can be identified by RFP expression in their eyes, and females produce embryos in which Bcd is distributed in a gradient. Males from this transgenic stock are crossed to females expressing a heat shock inducible flippase ( hsFLP) , and heat shocking the F1 larvae results in recombination and excision of the cassette at the FRT sites, bringing the sqh 3'UTR directly downstream of the bcd coding sequence. This initially results in mosaic F1 flies with a mosaic graded/uniform Bcd germline. The F1 are further outcrossed to bcd E1 mutants and F2 individuals producing embryos with uniform Bcd distributions can be identified by the lack of RFP expression in the eyes. ( D ) Expression levels of uniform Bcd constructs measured by western blots. Western blots for GFP-Bcd were performed on embryos at NC14. Representative gels and quantifications are shown for the bcd promoter-driven transgene ( A ), mtrm promoter-driven transgene ( B ) and αTub67C promoter-driven transgene ( C ). In the barplots, band intensities are reported relative to wild-type (GFP-Bcd). All lanes are normalized to an α-tubulin loading control. Error bars are standard deviation between two biological replicates for each sample. MW = molecular wt marker, *=skipped lane.

Article Snippet: A sequence containing the bcd 3'UTR and a 3xP3-RFP reporter flanked by FRT sites was synthesized by GenScript and cloned by Gibson Assembly into the pBABR GFP-Bcd3'sqh plasmid.

Techniques: Mutagenesis, Expressing, Clone Assay, Confocal Microscopy, Fluorescence, Generated, Transformation Assay, Binding Assay, Sequencing, Transgenic Assay, Construct, Western Blot, Control, Standard Deviation, Marker

Journal: Autophagy

Article Title: Astrocytes promote progression of breast cancer metastases to the brain via a KISS1-mediated autophagy

doi: 10.1080/15548627.2017.1360466

Figure Lengend Snippet: KISS1 expression correalates with p62/SQSTM1. (A) KISS1 expression reverses autophagy induction in the model of MDA-MB-231Br cells lacking KISS1 expression (MDA-MB-231Br-KISS1KD). Western blotting assessment of ATG7, LC3, ATG5, SQSTM1 and human GAPDH during KISS1 infection by selfreplicating oncolytic adenovirus. Experiment was conducted twice and data from one experiment are shown. (B and C) KISS1 knockdown promotes autophagy in MDA-MB-231Br cells. (B) Lentivirus-transduced MDA-MB-231Br cell lines shScrambled or KISS-KD61 were pretreated with DMSO, 1 mM of 3-Ma or 1 nM of BafA1 for 4 h and cell lysates were prepared and analyzed by western blots 48 h later using 10 µg of total protein loaded on each lane and the membrane was incubated with SQSTM1- or GAPDH-specific antibodies. (C) Ad or Ad-KISS1-transduced MDA-MB-231Br cells were pretreated with DMSO, 1 mM of 3-Ma or 1 nM of BafA1 for 4 h and cell lysates were prepared and analyzed by western blots 48 h later using 10 µg of total protein loaded on each lane and the membrane was incubated with SQSTM1- or GAPDH-specific antibodies. (D to F) Representative IHC or immunofluorescence images of brain metastatic BrCa (RONC-BM41 and RONC-BM23) human tissue specimens (D, E) or mouse-implanted MDA-MB-231Br human intracranial xenografts (F) stained for SQSTM1 and KISS1 demonstrate a direct correlation between KISS1 and SQSTM1 gene expression. IHC staining intensity for KISS1 (graded as 0 and 1+) showed direct correlation with that for SQSTM1 (graded as 1+ and 2+) in RONC-BM41 and RONC-BM23, respectively (D). Original image magnifications are 200X (scale bar: 50 µm) and 400X (scale bar: 20 µm); expression of SQSTM1 and KISS1 was visualized using brown dye DAB, conjugated to secondary antibody with background staining with blue dye haematoxylin or using an avidin-biotin conjugation system (F). A direct correlation between expression of SQSTM1 and KISS1 was also validated by RT-qPCR analysis of the corresponding gene's mRNA expression (E) as well as IF staining of murine intracranial MDA-MB-231Br xenografts (MDA231Br ic, (F) counterstained with DAPI using human KISS1- and SQSTM1-specific antibodies. White arrows indicate BrCa cells with detectable KISS1 (red signal) expression that in most cells colocalizes with SQSTM1 (green signal) producing orange foci (white arrows) on the merged (400X) image; scale bar: 20 µm. Spearman correlation test between KISS1 and SQSTM1 mRNA expression was used to analyze the mRNA expression results (E) (r = 0.99, P = 0.015).

Article Snippet: A) CN34Br or MDA-MB-231Br cells were labeled with premade lentiviruses expressing codon optimized RFP reporter under control of the CMV promoter (GenTarget Inc., LVP023).

Techniques: Expressing, Western Blot, Infection, Knockdown, Membrane, Incubation, Immunofluorescence, Staining, Gene Expression, Immunohistochemistry, Avidin-Biotin Assay, Conjugation Assay, Quantitative RT-PCR

Journal: Autophagy

Article Title: Astrocytes promote progression of breast cancer metastases to the brain via a KISS1-mediated autophagy

doi: 10.1080/15548627.2017.1360466

Figure Lengend Snippet: MIR-345–5p expression opposes KISS1 in breast cancer cells and primary samples. (A) Relative expression of KISS1 mRNA following transfection of MDA-MB-231 or MDA-MB-231Br cells with either nontargeting miRNA (red bars) or MIR345 mimic (blue bars) precursors as determined by RT-qPCR. (B) Relative expression of a Luciferase gene reporter with (blue bars) or without (red bars) the hKISS1−3′UTR sequence genetically fused downstream of the reporter-coding sequence in MDA-MB-231Br cells upon their cotransfection with MIR345 mimic or nontargeting control miRNA. The hKISS1−3′UTR reporter fusion was under control of CMV promoter. Luciferase activity of samples was normalized to that of negative control and data presented as a relative fold difference ± SD. Data represent means of 2 independent experiments performed in triplicate; P < 0.05 indicates statistical significance determined by a Student t test. (C) Inverse correlation between expression of MIR345–5p and KISS1 mRNA levels in human brain metastatic specimens as determined by RT-qPCR and the Pearson correlation test; Pearson coefficient r = −0.67, P = 0.0062; blue bars, KISS1 mRNA copy number; red bars, human MIR345 copy number both normalized by ACTB and RNU6–2 housekeeping RNA. (D) A Kaplan-Meier survival curve for brain metastatic patients expressing either high or low levels of MIR345–5p (MIR345) as determined by RT-qPCR in the paraffin-embedded tissue samples. Statistical analysis is performed by a Log Rank Test, P = 0.017.

Article Snippet: Infection of BrCa cells list-behavior=simple prefix-word= mark-type=none max-label-size=2 A) CN34Br or MDA-MB-231Br cells were labeled with premade lentiviruses expressing codon optimized RFP reporter under control of the CMV promoter (GenTarget Inc., LVP023).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Luciferase, Sequencing, Cotransfection, Control, Activity Assay, Negative Control