Journal: eLife

Article Title: Supramammillary nucleus synchronizes with dentate gyrus to regulate spatial memory retrieval through glutamate release

doi: 10.7554/eLife.53129

Figure Lengend Snippet:

Article Snippet: Antibody , Goat polyclonal Anti- GPF , Rockland , Cat# 600-101-215; RRID: AB_218182 , 1:1 k.

Techniques: Virus, Plasmid Preparation, Software

Journal: Oncoscience

Article Title: MDA-9/Syntenin regulates differentiation and angiogenesis programs in head and neck squamous cell carcinoma.

doi: 10.18632/oncoscience.99

Figure Lengend Snippet: Figure 4: Effect of MDA-9/Syntenin depletion on tumor growth and differentiation of HNSCC cells. (A) MDA-9/Syntenin depleted HNSCC xenografts exhibited decreased tumor growth (p=0.01-0.02). (B) Empty vector treated cells appeared poorly differentiated with more nuclear pleomorphism, larger nucleoli, and more clumped chromatin in vivo (left panel, black arrowheads) compared to the MDA-9/Syntenin depleted cells, which appeared moderately differentiated (right panel, red arrowheads). (C) Under in vitro condition, MDA-9/Syntenin depleted SCC-15 and FaDu cells also appeared more differentiated compared to the empty vector treated cells (right panel, red- and yellow arrowheads). Magnification X 200. (D-E) Restored expression of SPRR1B in vitro in all but one MDA-9/Syntenin depleted HNSCC cell lines (D) and SCC-15 xenograft (E). (F) SPRR1B expression was low or barely detectable by Western blotting in primary HNSCC tumor positive for lymph node metastasis (Table S1). Actin was used as loading control.

Article Snippet: HEK293 cells were stably transfected with a GPF tagged SPRR1B plasmid (pCMV6-AC-GFP, # RG220108, Origene Inc.).

Techniques: Plasmid Preparation, In Vivo, In Vitro, Expressing, Western Blot, Control

Journal: Oncoscience

Article Title: MDA-9/Syntenin regulates differentiation and angiogenesis programs in head and neck squamous cell carcinoma.

doi: 10.18632/oncoscience.99

Figure Lengend Snippet: Figure 5: MDA-9/Syntenin regulation of SPRR1B. (A) Stable overexpression of MDA-9/Syntenin in HEK293 cells (HEK293- MDA-9) markedly decreased SPRR1B expression compared to control (HEK293- Vector), but stable overexpression of SPRR1B in H3K293 cells (HEK293-SPRR1B) did not alter MDA-9/Syntenin expression compared to control (HEK293-Vector). SPRR1B expression was elevated following stable depletion of MDA-9 from HEK293 cells (HEK293-Sh-MDA-9) compared to control (HEK293-Vector). Overexpression of MDA-9/Syntenin in HEK293 cells stably overexpressing SPRR1B (HEK293B-MDA-9) reduced SPRR1B expression compared to the corresponding empty vector control (HEK293-Vector). Actin was used as loading control. (B) FACS analysis of the GFP- tagged SPRRB1 overexpressing HEK293 cells (denoted as HEK293B) revealed marked loss of GFP positive cells following overexpression of MDA-9/Syntenin compared to the empty vector transfected cells (32.8% vs. 99.7%). (C) The map of the various deletion constructs and the wild type MDA-9/Syntenin used to transfect HEK293 cells. (D) Overexpression of the wild type MDA-9/Syntenin or domains containing N-terminal plus PDZ1 or PDZ1 plus PDZ2 reduced SPRR1B expression in the HK293 cells as indicated. Actin was used as loading control.

Article Snippet: HEK293 cells were stably transfected with a GPF tagged SPRR1B plasmid (pCMV6-AC-GFP, # RG220108, Origene Inc.).

Techniques: Over Expression, Expressing, Control, Plasmid Preparation, Stable Transfection, Transfection, Construct

Journal: bioRxiv

Article Title: RNase L reprograms translation by widespread mRNA turnover escaped by antiviral mRNAs

doi: 10.1101/486530

Figure Lengend Snippet: (A) IF for SG-associated proteins G3BP1 and PABPC1 in WT and RL-KO U-2 OS cells eight-hours post-transfection of poly(I:C) (500-ng/ml). Nuclei were stained with DAPI (blue). (B) G3BP1-positive foci binned by volume in WT and RL-KO from greater than 30 cells in 3 fields of U-2 OS cells. (C) IF for G3BP1 and PABPC1 in WT and RL-KO A549 cells 8 hours post-poly(I:C). Images for G3BP1 and PABPC1 staining are shown in Figure S1D. (D) Schematic of approach for RLBs purification. (E) Proteins identified by RLB mass spectrometry analysis and overlap with sodium arsenite-induced SG proteome (Data File S1). (F) G3BP1 and PABPC1 IF in PKR-KO and PKR and RNase L double KO (PKR/RL-KO) A549 cells rescued with RNase L (RL) or RNase L-R667A (RL-CM) six hours post-poly(I:C). Scale bars represent 15 μm.

Article Snippet: To generate the pLJM1-GFP-G3BP1 lentiviral vector, the GPF-G3BP1 coding sequence was subcloned from the pEGFP-C1-G3BP1+stop vector, a gift from Dr. Nancy Kedersha, via digestion with Nhe1 and EcoR1 and ligation into the pLJM1-EGFP lentiviral vector (Addgene: 19319) using T4 DNA ligase (New England BioLabs).

Techniques: Transfection, Staining, Purification, Mass Spectrometry

Journal: bioRxiv

Article Title: RNase L reprograms translation by widespread mRNA turnover escaped by antiviral mRNAs

doi: 10.1101/486530

Figure Lengend Snippet: (A) smFISH for AHNAK mRNA in WT and RL-KO U-2 OS cells +/− poly(I:C) (500-ng/ml) for two or six hours with G3BP1 as a RLBs/SG marker. N.r. indicates non-responsive cells with respect to RLB/SG assembly. (B) Quantification of AHNAK mRNA smFISH from (A), with analysis of 3 fields of 17-30 RLB/SG cells. (C) RT-qPCR analysis of AHNAK mRNA in WT and RL-KO A549 cells at zero- and six-hours post-poly(I:C) transfection. (D) smFISH for GAPDH mRNA (red) in WT and RL-KO U-2 OS cells two hours post-poly(I:C). (E) Quantification of GAPDH mRNA smFISH. (F) qRT-PCR quantification of GAPDH, Actin B, and Tubulin A mRNAs in WT and RL-KO A549 cells transfected with or without (Mock) poly(I:C) for six hours. Bars represent the average Ct value +/−S.E.M. from at least five independent experiments. (G) Oligo(dT) (red) and G3BP1 (green) staining of WT and RL-KO A549 cells two hours post-poly(I:C). Quantification of mean signal in SG+ cells shown in Figure S4J. Scale bars represent 15 μm.

Article Snippet: To generate the pLJM1-GFP-G3BP1 lentiviral vector, the GPF-G3BP1 coding sequence was subcloned from the pEGFP-C1-G3BP1+stop vector, a gift from Dr. Nancy Kedersha, via digestion with Nhe1 and EcoR1 and ligation into the pLJM1-EGFP lentiviral vector (Addgene: 19319) using T4 DNA ligase (New England BioLabs).

Techniques: Marker, Quantitative RT-PCR, Transfection, Staining

Journal: bioRxiv

Article Title: RNase L reprograms translation by widespread mRNA turnover escaped by antiviral mRNAs

doi: 10.1101/486530

Figure Lengend Snippet: (A) smFISH/IF for IFN-β mRNA (green) GAPDH mRNA (red) and IF for G3BP1 (cyan). Nuclei were stained with DAPI (gray). WT and RL-KO A549 cells +/− poly(I:C) with smFISH/IF performed six hours post-transfection. Cells that contain RLBs/SGs and IFN-β mRNA indicated by white arrows. Cells with RLBs/SGs without IFN-β mRNA are indicated by yellow arrows. Cells with IFN-β mRNA without RLBs/SGs are indicated by orange arrows. Cells lacking both RLBs/SGs and IFN-β mRNA indicated by blue arrows. Scale bars represent 15 μm. (B) RT-qPCR analysis of IFN-β, IL-6, and GADD34 mRNA expression in WT and RL-KO A549 cells six-hours post-transfection of poly(I:C). Bars represent the average Ct value +/− the S.E.M from greater than 5 independent experiments.

Article Snippet: To generate the pLJM1-GFP-G3BP1 lentiviral vector, the GPF-G3BP1 coding sequence was subcloned from the pEGFP-C1-G3BP1+stop vector, a gift from Dr. Nancy Kedersha, via digestion with Nhe1 and EcoR1 and ligation into the pLJM1-EGFP lentiviral vector (Addgene: 19319) using T4 DNA ligase (New England BioLabs).

Techniques: Staining, Transfection, Quantitative RT-PCR, Expressing