Journal: bioRxiv

Article Title: Detailed analysis of chick optic fissure closure reveals Netrin-1 as an essential and conserved mediator of epithelial fusion during vertebrate embryogenesis

doi: 10.1101/477729

Figure Lengend Snippet: ( a ) Whole mounted embryos from HH.St25, and HH.St30 illustrated the optic fissure margin (OFM; arrows) as a non-pigmented region in the ventral aspect of the developing eye. ( b ) Flat-mounted ventral eye tissues revealed fusion dynamics during closure. At HH.St29 the medial OFM had narrowed markedly along the anterior-posterior (A-P) axis between the iris and the posterior region and fusion plate 1 (FP1) and FP2 (arrowheads) are adjacently positioned in the anterior OFM. At HH.St31 the medial OFM had become fully pigmented in the fused seam, and the distance between FP1 and FP2 (arrowheads) had lengthened in the A-P axis (hatched arrows). An opening remained at the anterior region of the iris (asterisk). ( c ) Fluorescent confocal microscopy of memGFP embryos was used to unambiguously define fusion plates (arrowheads) and seam throughout all stages, supported by bright-field microscopy. All fissures were also serially-sectioned to confirm the location of each fusion plate ( Supplemental Table S1 ). ( d ) Schematic representation of chick OFC progression in the anterior and medial retina. 1. Pre-fusion : A fully open OFM is evident in the ventral retina at stages HH.St25-27; 2. Initiation : At HH.St27-28 the first fusion is observed in the anterio-medial OFM; 3. Active fusion : fusion extends briefly in the anterior direction but then stops in the presumptive iris to leave an open region throughout development. Fusion proceeds markedly posteriorly with FP2 extending towards the pecten. 4. Complete fusion : Fusion stops posteriorly when FP2 meets the fused pecten region. The fusion seam is complete and epithelial remodelling occurs to generate a complete continuum of both NR and RPE. Abbreviations: ln, lens; r, retina, OF, optic fissure; A-P, anterior-posterior; FP, fusion point; HH, Hamburger Hamilton staging; RPE, retinal pigmented epithelia; NR, neural retina. ( e ) Phospho-Histone H3A immunostaining of serially-sectioned fissures at HH.St29-30 revealed the presence of mitotic cells within the apical neural retina throughout the ventral eye, but that PH3A was not enriched in the fused seam. ( f ) Quantified Phospho-Histone H3A immunostaining of whole-mounted fissures using confocal microscopy, confirmed that there was no significant enrichment for cell proliferation within the seam compared to equivalent nasal and temporal regions (Outside Seam) along the A-P axis.

Article Snippet: Antibodies were used against Phosphor-Histone H3A (Cell Signalling Technologies #33770 at 1:1000) and Netrin-1 (R&D Systems MAB128; 1:100).

Techniques: Confocal Microscopy, Microscopy, Immunostaining

Journal: bioRxiv

Article Title: Detailed analysis of chick optic fissure closure reveals Netrin-1 as an essential and conserved mediator of epithelial fusion during vertebrate embryogenesis

doi: 10.1101/477729

Figure Lengend Snippet: ( a ) Mean eye diameter measurements for chick embryonic days E4-E8 ( n ≥ 5 eyes per stage; one eye per embryo). ( b ) Location and orientation of the pecten oculi and associated blood vessel entering at the open iris fissure region at embryonic day 12. ( c ) Whole embryo and memGPF confocal images at HH.St25 and HH.St26 at anterior and midline illustrated the non-fused iris and medial fissure margin. ( d ) Representative H&E sections from fissures at HH.St25 and HH.St26 confirming lack of fusion at these stages. ( e ) Representative images of whole embryos and flat-mounted fissures from fusion-relevant Hamburger Hamilton , embryonic stages. The initiating plate (white arrow) is indicated for a HH.St28 fissure. A minimum of 3 fissures were examined by confocal light-microscopy to identify fusion points and then additional samples were processed by serial cryo-sectioning to confirm fusion plates and fused seams ( Supplemental Table S1 ). ( f ) Histogram to illustrate the entire fused seam length at each HH stage. ( g ) Left : Schema for quantifying PH3A foci within whole mounted fissures using a grid system, with a representative image showing positive nuclei. A-P axis is shown. Right : Histograms indicating number of PH3A-positive foci in HH.St29 and HH.St30 ventral eyes from whole mount Phospho-Histone H3A immunostaining to compare the fusion seam with non-seam regions in the ventral eye. Data shows mean PH3A+ cells per region at each stage. Note: seam length was too small to quantify in HH.St29 fissures, however PH3A foci were fewer at the fusion points compared to non-seam regions. Data shown is the mean of three fissures per stage with standard deviations indicated (error bars = 1x s.d.).

Article Snippet: Antibodies were used against Phosphor-Histone H3A (Cell Signalling Technologies #33770 at 1:1000) and Netrin-1 (R&D Systems MAB128; 1:100).

Techniques: Light Microscopy, Immunostaining

Journal: Nature Communications

Article Title: SMARCA4 -inactivating mutations increase sensitivity to Aurora kinase A inhibitor VX-680 in non-small cell lung cancers

doi: 10.1038/ncomms14098

Figure Lengend Snippet: ( a ) Cartoon showing that TPX2 and RAN act sequentially to activate AURKA, a known regulator of mitosis. RAN prevents the inhibitory interaction of Importins on TPX2, allowing it to activate AURKA for mitotic spindle assembly. ( b ) Immunoblot analysis confirms that three of the four siRNAs greatly reduce TPX2 protein levels in NCI-H1819 cell lysates 3 days after transfecting the cells. ( c ) Five days after transfecting NCI-H1819 cells with non-targeting or individual siRNAs targeting TPX2, cell viability was measured with a CellTiter-Glo viability assay. PLK1 was depleted as the positive control. NT=nontargeting. ** indicates statistical significance ( P <0.001). Statistical significance was assessed by one-way ANOVA and post hoc Dunnett's multiple comparison tests. ( d ) The effect of individual siRNAs on NCI-H1819 cells was measured by immunoblotting with monoclonal antibodies to cleaved poly (ADP-ribose) polymerase 1 (PARP), phospho-Histone H3 and β-Actin as a loading control 3 days after transfecting the cells with either non-targeting or TPX2-targeting siRNAs. Cleaved PARP indicated an active apoptotic response and phospho-Histone H3 indicated mitotic arrest. Data are representative of duplicate experiments. ( e ) Five days after transfecting NCI-H1819, NCI-1299, NCI-H23, HCC827 and Calu-1 cells with non-targeting or individual siRNAs #5 and #6 targeting TPX2, cell viability was measured with a CellTiter-Glo assay that measure cellular ATP as a surrogate for cell proliferation or survival. PLK1 was depleted as the positive control. Error bars on graphs are s.d. of means from triplicate biological replicates.

Article Snippet: TPX2 (Biolegend, mouse mAb, 628001, 1:1,000 dilution), AURKA (Cell Signaling, rabbit mAb, 4718, 1:1,000 dilution), Phospho-Histone H3 (Cell Signaling, rabbit mAb, 33770, 1:1,000 dilution), cleaved PARP (Cell Signaling, rabbit mAb, 9541, 1:1,000 dilution), FLAG (Cell Signaling, rabbit mAb, 2368, 1:1,000 dilution), SMARCA4/BRG1 (EMD Millipore, rat mAb, MABE60, 1:1,000 dilution), DLG7/HURP (Bethyl Laboratory, A300-852A, 1:1,000 dilution), c-MYC (Santa Cruz Biotechnology, mouse mAb, sc-40, 1:1,000 dilution) and β-Actin (MP Biomedicals, mouse mAb, 69100, 1:10,000 dilution) antibodies were used for immunoblotting assays.

Techniques: Western Blot, Viability Assay, Positive Control, Comparison, Bioprocessing, Control, Glo Assay

Journal: Nature Communications

Article Title: SMARCA4 -inactivating mutations increase sensitivity to Aurora kinase A inhibitor VX-680 in non-small cell lung cancers

doi: 10.1038/ncomms14098

Figure Lengend Snippet: ( a ) NCI-H1819 cell lysates were collected 3 days after transfecting the cells with either non-targeting or AURKA-targeting siRNAs and were immunoblotted to monitor AURKA protein levels. ( b ) Five days after transfecting NCI-H1819 cells with individual siRNAs, cell viability was measured with a CellTiter-Glo assay on triplicate biological replicates. PLK1 knockdowns were used as the positive control. Means with s.d. are shown on the graphs. * indicates statistical significance ( P <0.001) and ** indicates statistical significance ( P <0.001) combined with higher biological significance (viability<50%). ( c ) The response to depletion of AURKA was assessed by immunoblotting with monoclonal antibodies to cleaved PARP, phospho-Histone H3 and β-Actin. ( d ) Five days after depleting AURKA in NCI-H1819, NCI-H1819-pBABE and NCI-H1819-pBABE-SMARCA4-FLAG cells, cell viability was measured with a CellTiter-Glo assay as in part ( b ). ( e ) Five days after transfecting NCI-H1819, NCI-1299, NCI-H23, HCC827 and Calu-1 cells with non-targeting or individual siRNA #28 targeting AURKA, cell viability was measured with a CellTiter-Glo viability assay as in . PLK1 was depleted as the positive control. Error bars on graphs are s.d. of means from triplicate biological replicates. Statistical significance on graphs was assessed by one-way ANOVA and post hoc Dunnett's multiple comparison tests.

Article Snippet: TPX2 (Biolegend, mouse mAb, 628001, 1:1,000 dilution), AURKA (Cell Signaling, rabbit mAb, 4718, 1:1,000 dilution), Phospho-Histone H3 (Cell Signaling, rabbit mAb, 33770, 1:1,000 dilution), cleaved PARP (Cell Signaling, rabbit mAb, 9541, 1:1,000 dilution), FLAG (Cell Signaling, rabbit mAb, 2368, 1:1,000 dilution), SMARCA4/BRG1 (EMD Millipore, rat mAb, MABE60, 1:1,000 dilution), DLG7/HURP (Bethyl Laboratory, A300-852A, 1:1,000 dilution), c-MYC (Santa Cruz Biotechnology, mouse mAb, sc-40, 1:1,000 dilution) and β-Actin (MP Biomedicals, mouse mAb, 69100, 1:10,000 dilution) antibodies were used for immunoblotting assays.

Techniques: Glo Assay, Positive Control, Western Blot, Bioprocessing, Viability Assay, Comparison

Journal: Nature Communications

Article Title: SMARCA4 -inactivating mutations increase sensitivity to Aurora kinase A inhibitor VX-680 in non-small cell lung cancers

doi: 10.1038/ncomms14098

Figure Lengend Snippet: ( a ) The response to depleting HURP with a pool of four individual siRNAs was measured with monoclonal antibodies to HURP, cleaved PARP (indicating apoptosis) and phospho-Histone H3 (indicating cells engaged in mitosis). ( b ) Five days after depleting HURP in NCI-H1819, NCI-H1819-pBABE and NCI-H1819-pBABE-SMARCA4-FLAG cells and ( c ) in NCI-H1819, NCI-1299, NCI-H23, HCC827 and Calu-1 cells, cell viability was measured with a CellTiter-Glo assay as in and . PLK1 was depleted as the positive control. Data are means with s.d. of triplicate biological replicates and representative of triplicate experiments. ( d ) Cell lysates of NCI-H1819, NCI-H1819-pBABE and NCI-H1819-pBABE-SMARCA4-FLAG cells were immunoblotted with monoclonal antibodies to HURP. ( e ) HURP expression was measured in a panel of SMARCA4 -mutant or wild-type NSCLCs and HBECs by immunoblotting. ( f ) HURP protein band intensities were measured with the ImageQuant software and graphed. Horizontal bars indicate means and s.e.m. of sample groups. Statistical significance on graphs was assessed by one-way ANOVA and post hoc Dunnet's multiple comparison tests.

Article Snippet: TPX2 (Biolegend, mouse mAb, 628001, 1:1,000 dilution), AURKA (Cell Signaling, rabbit mAb, 4718, 1:1,000 dilution), Phospho-Histone H3 (Cell Signaling, rabbit mAb, 33770, 1:1,000 dilution), cleaved PARP (Cell Signaling, rabbit mAb, 9541, 1:1,000 dilution), FLAG (Cell Signaling, rabbit mAb, 2368, 1:1,000 dilution), SMARCA4/BRG1 (EMD Millipore, rat mAb, MABE60, 1:1,000 dilution), DLG7/HURP (Bethyl Laboratory, A300-852A, 1:1,000 dilution), c-MYC (Santa Cruz Biotechnology, mouse mAb, sc-40, 1:1,000 dilution) and β-Actin (MP Biomedicals, mouse mAb, 69100, 1:10,000 dilution) antibodies were used for immunoblotting assays.

Techniques: Bioprocessing, Glo Assay, Positive Control, Expressing, Mutagenesis, Western Blot, Software, Comparison