Journal: Oncotarget

Article Title: A novel lncRNA, GASL1, inhibits cell proliferation and restricts E2F1 activity

doi: 10.18632/oncotarget.15864

Figure Lengend Snippet: (A) U2OS cells were transfected with non specific siRNA (NS) or two distinct GASL1 -specific siRNAs (si GASL1 #1 or #2) and cells were analyzed by FACS 72h post-transfection. Percentages of cells in G1, S, and G2/M cell-cycle phases are depicted. Results shown are an average of six independent experiments (*p<0.05,**p<0.01, ***p<0.005; two-tailed Student's t-test). (B) U2OS cells were transfected with either a nonspecific siRNA (NS) or siRNA directed against GASL1 . After 48 hr, cells were left untreated or incubated with Hydroxyurea (4mM, 18hr). The cells were then allowed to resume growth for 4 or 6 hours in fresh media. Cells were analyzed by FACS using Propidium-Iodide (PI) staining. (C) The bar graphs depict the average cell cycle distribution from four independent experiments in which cells were treated as in C (*p< 0.05, **p<0.01; two-tailed Student's t-test).

Article Snippet: For transfecting U20S cells with plasmids, polyjet transfection reagent (Signagen) was employed according to the manufacturer's instructions.

Techniques: Transfection, Two Tailed Test, Incubation, Staining

Journal: Oncotarget

Article Title: A novel lncRNA, GASL1, inhibits cell proliferation and restricts E2F1 activity

doi: 10.18632/oncotarget.15864

Figure Lengend Snippet: (A) U2OS cells were transfected with siRNA directed against GASL1 or a non specific siRNA (NS). Total RNA was extracted from the cells and the level of Gasl1 RNA was determined by real time qPCR, and normalized to Gapdh . (B) U2OS cells were transfected with either a non specific siRNA (NS) or GASL1 -specific siRNA and cells were counted at the indicated times post transfection. The cell numbers shown are an average of three independent experiments, each performed in triplicates. (C) U2OS cells stably expressing shRNA against GASL1 or a non specific shRNA were seeded at 5000 cells/dish, and cultured for 3 weeks. Then, the plates were Giemsa stained and the number of colonies determined. The assay was performed in triplicates. (D) The average numbers of colonies of four independent experiments performed as described in (C) (*p<0.05. two-tailed Student's t-test)

Article Snippet: For transfecting U20S cells with plasmids, polyjet transfection reagent (Signagen) was employed according to the manufacturer's instructions.

Techniques: Transfection, Stable Transfection, Expressing, shRNA, Cell Culture, Staining, Two Tailed Test

Journal: Frontiers in Physiology

Article Title: Pathogenic gain-of-function mutations in the prodomain and C-terminal domain of PCSK9 inhibit LDL binding

doi: 10.3389/fphys.2022.960272

Figure Lengend Snippet: Ser-127 residue is critical to LDL binding function of PCSK9. (A) 24 h post-transfection, HEK293 cells transiently expressing WT-PCSK9 or indicated S127 mutants were harvested and cell lysates and medium immunoprecipitates were analyzed by 8% SDS-PAGE and western blotting to detect precursor (P) and mature (M) forms of PCSK9. # indicates a faster-migrating form of PCSK9 consistent with furin-mediated proteolysis. Shown is a representative experiment ( n = 3) (B) Mouse Hepa-1c1c7 cells cultured in sterol-depleting medium were treated for 4 h with WT or indicated mutant forms of PCSK9 (2.5 μg/ml). Biotinylated cell surface LDLRs were isolated and quantified by Western blotting using transferrin receptor (TfR) as a loading control. Shown are representative western blots ( top ) with densitometric analysis of three independent experiments ( bottom ). Error bars represent SEM ( n = 3). Significant change in LDLR expression compared to WT-PCSK9 was determined by Student’s t -test: ** p < 0.01. (C) Conditioned cell culture medium containing WT-PCSK9 (WT) or indicated mutants were incubated with LDL prior to density gradient-ultracentrifugation and immunoprecipitation and Western blot analysis of LDL-containing fractions. Shown is a representative experiment ( n = 3)

Article Snippet: We obtained fetal bovine serum (FBS) and newborn calf serum from ThermoFisher, EDTA-free Complete™ Protease Inhibitor Tablets were from Roche, Optiprep™ density gradient medium (60% w/v iodixanol) from Axis-Shield, NP-40 detergent was from Biovision and PolyJet DNA transfection reagent was from FroggaBio (Toronto, Ontario).

Techniques: Binding Assay, Transfection, Expressing, SDS Page, Western Blot, Cell Culture, Mutagenesis, Isolation, Incubation, Immunoprecipitation

Journal: Molecular Systems Biology

Article Title: Enhancers and genome conformation provide complex transcriptional control of a herpesviral gene

doi: 10.1038/s44320-024-00075-0

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Polyjet In Vitro Transfection Reagent , SignaGen , NC1536117.

Techniques: Recombinant, Sequencing, Library Quantification, SYBR Green Assay, cDNA Synthesis, Purification, Gel Extraction, In Vitro, Transfection, Protease Inhibitor, Software