Journal: PLoS ONE

Article Title: Targeting mitochondrial DNA polymerase gamma for selective inhibition of MLH1 deficient colon cancer growth

doi: 10.1371/journal.pone.0268391

Figure Lengend Snippet: Real-time dynamic monitoring of the cytotoxic effects of CR on (A) HCT116VA (MLH1 deficient) (B) HCT116V1 (MLH1 proficient) (C) Lovo (MLH1 proficient) (D) MCF7 (MLH1 proficient) cells using the xCELLigence system. Cell growth was continuously monitored every 30 min. Cell index was normalized to the time point of CR administration. Normalized cell index was plotted as the mean value from triplicates; error bars represent the standard deviation of the mean. The Grey arrow indicates the time of CR administration. The xCELLigence RTCA software was used to determine IC 50 values at 48h post-treatment time point. (E) Effect of 5 μM CR (IC 50 = 5.19 μM) on colony formation in HCT116VA and HCT116V1 cells. Data are expressed as the mean ± standard deviation of three independent experiments. (F) Upper panel, Real-time dynamic monitoring of the cytotoxic effects of CR on CRISPR/Cas9 Pol γ knockout HCT116 cells and lower panel HCT116 control cells (empty vector control). (G) Real-time dynamic monitoring of the cytotoxic effects of CR on shRNA Pol γ knockdown HCT116V1 cells and HCT116V1 control cells.

Article Snippet: HCT116V1 cells were transfected with 50 nM shRNA targeting Pol γ catalytic subunit (OriGene, cat no.TR310307) and control shRNA (OriGene, cat no. TR30012) using FuGENE HD transfection reagent (Promega) according to the manufacturer’s instructions.

Techniques: Standard Deviation, Software, CRISPR, Knock-Out, Plasmid Preparation, shRNA

Journal: The Journal of Biological Chemistry

Article Title: Folic Acid Remodels Chromatin on Hes1 and Neurog2 Promoters during Caudal Neural Tube Development *

doi: 10.1074/jbc.M110.126714

Figure Lengend Snippet: KDM6B directly regulates histone H3K27 methylation in WT cultured neurospheres. A, transfection of scrambled negative control shRNA-RFP (TR30015, OriGene) into cells from WT neurospheres and immunostaining for KDM6B; B, transfection with KDM6B shRNA-RFP and immunostaining for KDM6B; C, transfection of scrambled negative control shRNA-RFP and immunostaining for H3K27me3; D, transfection with KDM6B shRNA-RFP and immunostaining for H3K27me3. Experiments were performed in quadruplicate. A representative of 4 separate experiments is shown. These results demonstrate that H3K27 methylation is directly regulated by KDM6B in cultured neurospheres.

Article Snippet: A scrambled negative control-shRNA-RFP from OriGene (TR30015) did not silence KDM6B levels ( A ) or increase H3K27 methylation ( C ).

Techniques: Methylation, Cell Culture, Transfection, Negative Control, shRNA, Immunostaining

Journal: Frontiers in Molecular Biosciences

Article Title: LncRNA NEAT1 Promotes High Glucose-Induced Mesangial Cell Hypertrophy by Targeting miR-222-3p/CDKN1B Axis

doi: 10.3389/fmolb.2020.627827

Figure Lengend Snippet: NEAT1 promotes HG-induced hypertrophy. (A) Expression of NEAT1 in renal tissues of patients with diabetic nephropathy (DN, n = 30) versus controls (n = 30) determined by Real-time PCR. Human mesangial cells were treated with 5 mM glucose and 20 mM mannitol (NG) or 25 mM glucose (HG); (B) NEAT1 expression was measured by Real-time PCR in cells treated with HG for indicated times. (C-H) Cells were transfected with control vector or NEAT1 shRNA, and cells were treated with NG or HG for 24 h; (C) Expression of NEAT1 was measured by Real-time PCR; (D, E) Distribution of cells in different stages of cell cycle was quantified by FACS; (F) Protein synthesis was measured by quantifying the incorporation of 35 S-methionine; (G) Hypertrophy was measured by quantifying average protein content per cell; (H) Expression of NEAT1 was measured by Real-time PCR. * indicates P <0.05, *** indicates P <0.001 compared with control or NG, # indicates P<0.05, ### indicates P <0.001 compared with HG + shNC. Data were expressed as mean ± SD, and experiments were repeated three times.

Article Snippet: The NEAT1 shRNA (shNEAT1#1, 5′-GTC TGT GTG GAA GGA GGA A-3′; shNEAT1#2, 5′-TGG AGG AGT CAG GAG GAA T-3′; shNEAT1#3, 5′-GAG GAG TCA GGA GGA ATA G-3′) or scramble shRNA control (shNC, 5′-GTA GAG TCA GCG AGA ATC T-3′) sequences were designed and synthesized by Sangon Biotech (Shanghai, China), and subcloned into pLKO.1 (Addgene, Watertown, MA, United States).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Control, Plasmid Preparation, shRNA