Journal: Nucleic Acids Research

Article Title: HCR-Proxy resolves site-specific proximal RNA microenvironments at subcompartmental resolution

doi: 10.1093/nar/gkag086

Figure Lengend Snippet: Spatially resolved rRNA-proximal proteomes within subnucleolar compartments. ( A ) Schematics of probes designed for HCR-Proxy MS to spatially resolve pre-rRNA proteomes within multiphased nucleoli. ( B ) HCR-FISH deconvolved STED micrographs of probes targeting distinct regions of the nascent rRNA transcript (magenta, A′ UP; yellow, A′ DOWN; blue, ITS2). ( C and D ) Dot plots displaying enriched subnucleolar HCR-Proxy components, demonstrating the ability of HCR-Proxy MS to spatially resolve multiphased nucleolar interactomes between (C) A′ UP and A′ DOWN, and (D) A′ UP and the TS2 region, respectively. Error bars present the SDs. ( E ) Volcano plot of enriched interactors specific for A′ UP region (magenta), its newly validated interactor (green) and A′ DOWN region (yellow). The cut-off for identification of bona fide interactors was set on LASSO regularization-based log2 fold-change > 1 and associated −log10( P -value) > 1. For visualization purposes, −log10( P ) values ( y -axis) were thresholded at 5 (dashed line). ( F ) HCR-FISH IF of the newly validated A′ UP interactor. RNA FISH (magenta, A′ UP; yellow, A′ DOWN) and newly validated nucleolar protein localized to FC (grey, TXNRD1). Quantification of the signal co-localization between RNA target (magenta, A′ UP; yellow, A′ DOWN) and candidate protein TXNRD1 with the correlation analysis; direct image correlation at zero displacement (i.e. overlap, left), and displacement-dependent correlation (right; C > 1 indicates above-random co-localization; see the Materials and methods for details). Each dot or thin curve represents one of the ~10 images per condition acquired, the bands represent 80% confidence intervals, and thick curves the mean values (one-way Mann–Whitney test; *** P < 0.001, ** P < 0.01, * P < 0.05).

Article Snippet: To validate co-localization with nuclear condensates and to confirm localization of candidate proteins, we used the following antibodies against: SC-35 (mouse, Santa Cruz Biotechnology, cat #sc-53518, 1:200 dilution), NPM1 (mouse, ThermoFisher Scientific, cat #32-5200, 1:100 dilution), FBL (rabbit, Abcam, cat #ab5821, 1:200 dilution), TXNRD1 (rabbit, Proteintech, cat #11117-1-AP, 1:200 dilution), and GFP (rabbit, ThermoFisher Scientific, cat #A-21311, 1:100 dilution).

Techniques: MANN-WHITNEY

Journal: Proteomics

Article Title: Proteome-wide changes induced by the Hsp90 inhibitor, geldanamycin in anaplastic large cell lymphoma cells.

doi: 10.1002/pmic.200700108

Figure Lengend Snippet: Figure 6. Immunoblot validation of differentially expressed pro- teins as identified by LC-MS/MS. Total cell lysate was extracted from control and GA-treated cells after 12 h and proteins sepa- rated by SDS-PAGE. Immunoblots were probed with anti-USP9 (1:1000), anti-GAP1 (1:300), anti-PCLN (1:300), anti-STCN (1:300), anti-tankyrase (1:1000), anti-SOCS-4 (1:1000), anti-NEMO (1:1000), anti-OPG (1:500), and anti-actin (1:500). Immunoblots were then incubated at room temperature for 1 h followed by probing with respective HRP-conjugated secondary antibodies and visualized by chemiluminescence. The left panels depict the overexpressed proteins as identified by LC-MS/MS in control and cells exposed to 10 mM GA. The right panels depict the under- expressed proteins as identified in the same samples. Both cICAT- and densitometry fold-change is listed below the 10 mM GA lanes. Actin served as a loading control.

Article Snippet: The following antibodies were used for immunoblot analysis: mouse polyclonal antibodies against PARP, p21, and p27; goat polyclonal antibodies against ras GTPase-activating protein 3 (RARS3), paracellin-1 (PCLN1), and stanniocalcin-1 (STCN1); and rabbit polyclonal antibody against actin from Santa Cruz Biotechnology (Santa Cruz, CA); mouse polyclonal antibodies against Hsp90 and NEMO (Becton Dickinson Pharmingen, San Diego); rabbit polyclonal antibodies against phospho-ALK and ALK (Cell Signaling, Beverly, MA); rabbit polyclonal antibody against ubiquitin-specific protease 9 (USP9) (Abgent, San Diego); mouse polyclonal antibodies against tankyrase (TNKS) and osteoprotegerin (OPG) and rabbit polyclonal antibodies against suppressor of cytokine signaling 4 (SOCS4; Acris Antibodies, Hiddenhausen, Germany).

Techniques: Western Blot, Biomarker Discovery, Liquid Chromatography with Mass Spectroscopy, Control, SDS Page, Incubation

Journal: PLoS ONE

Article Title: Recurrent rearrangements of the Myb/SANT-like DNA-binding domain containing 3 gene ( MSANTD3 ) in salivary gland acinic cell carcinoma

doi: 10.1371/journal.pone.0171265

Figure Lengend Snippet: (A) Predicted structure of the HTN3 - MSANTD3 fusion gene. Exon 1 (non-coding) of HTN3 is fused to the exon 2 (first coding exon) of MSANTD3 , leading to predicted overexpression of full-length MSANTD3 protein (translation start site is indicated). Fusion junction-spanning sequence reads are shown in . (B) Predicted structure of the PRB3 - ZNF217 gene fusion. Here, exon 2 (coding) of PRB3 is fused to exon 2 (first coding) of ZNF217 , possibly leading to the overexpression (by internal initiation of translation) of full-length ZNF217 protein. Fusion junction-spanning sequence reads are shown in .

Article Snippet: A human C-terminally Myc-tagged MSANTD3 cDNA ORF clone was obtained from Origene (SKU:RC203850), and then subcloned into pLentiCMV-Puro (Addgene #17452) to create pLentiCMV-MSANTD3. pLentiCMV-MSANTD3 or empty vector control was packaged in 293T cells (ATCC) using ViraPower Lentiviral Packaging Mix (Thermo Fisher Scientific), and then transduced into SMG-C6 cells and selected with 4 μg/ml puromycin.

Techniques: Over Expression, Sequencing

Journal: PLoS ONE

Article Title: Recurrent rearrangements of the Myb/SANT-like DNA-binding domain containing 3 gene ( MSANTD3 ) in salivary gland acinic cell carcinoma

doi: 10.1371/journal.pone.0171265

Figure Lengend Snippet: Acinic cell carcinoma cases.

Article Snippet: A human C-terminally Myc-tagged MSANTD3 cDNA ORF clone was obtained from Origene (SKU:RC203850), and then subcloned into pLentiCMV-Puro (Addgene #17452) to create pLentiCMV-MSANTD3. pLentiCMV-MSANTD3 or empty vector control was packaged in 293T cells (ATCC) using ViraPower Lentiviral Packaging Mix (Thermo Fisher Scientific), and then transduced into SMG-C6 cells and selected with 4 μg/ml puromycin.

Techniques:

Journal: PLoS ONE

Article Title: Recurrent rearrangements of the Myb/SANT-like DNA-binding domain containing 3 gene ( MSANTD3 ) in salivary gland acinic cell carcinoma

doi: 10.1371/journal.pone.0171265

Figure Lengend Snippet: (A-C) Break-apart FISH assay of MSANTD3 locus rearrangement; green FISH probe is flanking centromeric and red FISH probe is flanking telomeric to MSANTD3 . Note positive rearrangement (evidenced by physically-separated green and red signals, arrows) in AcCC case (A) . AcCC case (B) is negative for the rearrangement (i.e., signals co-localize), as is observed in normal salivary gland control (C) . (D) Break-apart FISH assay of ZNF217 rearrangement, confirming the rearrangement in the index case. (E-G) Corresponding MSANTD3 immunostaining of the above cases. Note the strong nuclear MSANTD3 staining (E) corresponding to the FISH-positive rearranged specimen.

Article Snippet: A human C-terminally Myc-tagged MSANTD3 cDNA ORF clone was obtained from Origene (SKU:RC203850), and then subcloned into pLentiCMV-Puro (Addgene #17452) to create pLentiCMV-MSANTD3. pLentiCMV-MSANTD3 or empty vector control was packaged in 293T cells (ATCC) using ViraPower Lentiviral Packaging Mix (Thermo Fisher Scientific), and then transduced into SMG-C6 cells and selected with 4 μg/ml puromycin.

Techniques: Immunostaining, Staining

Journal: PLoS ONE

Article Title: Recurrent rearrangements of the Myb/SANT-like DNA-binding domain containing 3 gene ( MSANTD3 ) in salivary gland acinic cell carcinoma

doi: 10.1371/journal.pone.0171265

Figure Lengend Snippet: MSANTD3 immunostaining of salivary gland benign and malignant specimens.

Article Snippet: A human C-terminally Myc-tagged MSANTD3 cDNA ORF clone was obtained from Origene (SKU:RC203850), and then subcloned into pLentiCMV-Puro (Addgene #17452) to create pLentiCMV-MSANTD3. pLentiCMV-MSANTD3 or empty vector control was packaged in 293T cells (ATCC) using ViraPower Lentiviral Packaging Mix (Thermo Fisher Scientific), and then transduced into SMG-C6 cells and selected with 4 μg/ml puromycin.

Techniques: Immunostaining

Journal: PLoS ONE

Article Title: Recurrent rearrangements of the Myb/SANT-like DNA-binding domain containing 3 gene ( MSANTD3 ) in salivary gland acinic cell carcinoma

doi: 10.1371/journal.pone.0171265

Figure Lengend Snippet: MSANTD3 immunostaining across diverse benign and neoplastic tissues.

Article Snippet: A human C-terminally Myc-tagged MSANTD3 cDNA ORF clone was obtained from Origene (SKU:RC203850), and then subcloned into pLentiCMV-Puro (Addgene #17452) to create pLentiCMV-MSANTD3. pLentiCMV-MSANTD3 or empty vector control was packaged in 293T cells (ATCC) using ViraPower Lentiviral Packaging Mix (Thermo Fisher Scientific), and then transduced into SMG-C6 cells and selected with 4 μg/ml puromycin.

Techniques: Immunostaining

Journal: PLoS ONE

Article Title: Recurrent rearrangements of the Myb/SANT-like DNA-binding domain containing 3 gene ( MSANTD3 ) in salivary gland acinic cell carcinoma

doi: 10.1371/journal.pone.0171265

Figure Lengend Snippet: (A) Schematic depiction of MSANTD3 domains, showing the location of the Myb/SANT-like domain within the N-terminus. (B) Schematic depiction of MSANTD3 MACAW sequence alignments across species. Boxes indicate conserved blocks, while the shading indicates pair-wise scores relative to human MSANTD3 with colors indicated in the key. Above , the horizontal black bar indicates the location of the conserved Myb/MSANT domain found by NCBI search (Pfam 13873). (C) Actual MACAW alignment within the MYB/SANT region. (D) Phylogenetic tree based on the global alignment made by ClustalX and visualized using Treeview software.

Article Snippet: A human C-terminally Myc-tagged MSANTD3 cDNA ORF clone was obtained from Origene (SKU:RC203850), and then subcloned into pLentiCMV-Puro (Addgene #17452) to create pLentiCMV-MSANTD3. pLentiCMV-MSANTD3 or empty vector control was packaged in 293T cells (ATCC) using ViraPower Lentiviral Packaging Mix (Thermo Fisher Scientific), and then transduced into SMG-C6 cells and selected with 4 μg/ml puromycin.

Techniques: Sequencing, Software

Journal: PLoS ONE

Article Title: Recurrent rearrangements of the Myb/SANT-like DNA-binding domain containing 3 gene ( MSANTD3 ) in salivary gland acinic cell carcinoma

doi: 10.1371/journal.pone.0171265

Figure Lengend Snippet: (A) Overexpression of C-terminally Myc-tagged MSANTD3 in SMG-C6 immortalized rat salivary gland epithelial cells, demonstrated by western blot using anti-MSANTD3 antibody ( left ) and anti-Myc tag antibody ( right ). Note, the observed band runs close to the calculated MW of the tagged protein (36 kD). (B) MSANTD3 overexpression does not enhance cell proliferation, compared to empty vector control, quantified by Wst-1 assay. (C) Transcriptome (RNA-seq) analysis of MSANTD3 overexpression identifies significant upregulation of gene sets associated with protein synthesis. Gene Set Enrichment Analysis (GSEA) of the top gene set (‘KEGG Ribosome’) is shown; other significant gene sets are listed in . Note, the early and positive upswing of the Enrichment profile reflects the early concentration of ribosome genes within the ranked list of genes upregulated by MSANTD3 overexpression (compared to empty vector control).

Article Snippet: A human C-terminally Myc-tagged MSANTD3 cDNA ORF clone was obtained from Origene (SKU:RC203850), and then subcloned into pLentiCMV-Puro (Addgene #17452) to create pLentiCMV-MSANTD3. pLentiCMV-MSANTD3 or empty vector control was packaged in 293T cells (ATCC) using ViraPower Lentiviral Packaging Mix (Thermo Fisher Scientific), and then transduced into SMG-C6 cells and selected with 4 μg/ml puromycin.

Techniques: Over Expression, Western Blot, Plasmid Preparation, WST-1 Assay, RNA Sequencing Assay, Concentration Assay