Journal: Cell Death and Differentiation

Article Title: FHL3 links cell growth and self-renewal by modulating SOX4 in glioma

doi: 10.1038/s41418-018-0152-1

Figure Lengend Snippet: FHL3 regulates the target genes SOX4, CAV1, and DDIT3 in glioma cells. a Glioma cell lines (T98G, U87MG, and U251) were transfected with PLVX empty vector (−) or FHL3 overexpression plasmid (+). Lysates were collected 48 h post-transfection and immunoblotted for the indicated proteins. β-Actin was used as a loading control. The bar graph shows cell viability relative to the control groups 96 h post-transfection. b Schematic illustration of the procedure used to screen and refine the set of FHL3-regulated target genes identified by three independent glioma microarray data replicates. c Twenty-eight indicated genes reported to be involved in glioma were assessed by microarray (gray bars) and real-time PCR (black bars). GAPDH was used as a housekeeping gene. d Heatmaps illustrating the expression profiles of the 11 differentially expressed genes verified by microarray experiments (n = 3 for each biological replicate). e Soluble chromatin was subjected to immunoprecipitation with IgG or anti-FHL3 antibodies in T98G cells. We designed two pairs of primers (P1 and P2) to amplify the predicted binding regions upstream of each gene, as peaks were identified in these regions in the ChIP-on-chip assay. The locations of the peaks identified by the ChIP-on-chip assay are denoted as short red bars. Immunoprecipitated DNA was PCR amplified with primers (locations indicated with short green bars) that annealed to the proximal region of the DDIT3, CAV1, or SOX4 promoters. The lengths of the amplified fragments are 247 bp (DDIT3-P1), 237 bp (DDIT3-P2), 263 bp (CAV1-P1), 219 bp (CAV1-P2), 288 bp (SOX4-P1), and 210 bp (SOX4-P2)

Article Snippet: Cell lines and cell culture The human glioma cell lines T98G, U87MG, and A172 were purchased from ATCC and cultured according to the guidelines recommended by the ATCC.

Techniques: Transfection, Plasmid Preparation, Over Expression, Microarray, Real-time Polymerase Chain Reaction, Expressing, Immunoprecipitation, Binding Assay, Amplification

Journal: Cell Death and Differentiation

Article Title: FHL3 links cell growth and self-renewal by modulating SOX4 in glioma

doi: 10.1038/s41418-018-0152-1

Figure Lengend Snippet: FHL3 inhibits glioma cell proliferation mainly through the downregulation of SOX4 expression. a Representative western blot showing CAV1, DDIT3, SOX4, and FHL3 protein levels in FHL3-overexpressing (+) glioma cell lines. b, c Western blot analysis of T98G, U87MG, and U251 glioma cell lines transfected with pcDNA6.0-Flag-CAV1 (b), pcDNA6.0-Flag-DDIT3 (c) or control vector (−). An anti-Flag antibody was used to detect target gene overexpression. Bar graphs show the results of MTS assays in the same three glioma cell lines 96 h after transfection with plasmids. d, e Western blot analysis of SOX4 knockdown and overexpression in T98G, U87MG, and U251 glioma cell lines following lentiviral infection with shSOX4 (d), LV-3Flag-SOX4 (e), or a control (−). Anti-SOX4 and anti-Flag antibodies were separately used to detect SOX4 knockdown and overexpression, respectively. Bar graphs show the results of MTS assays performed 96 h after lentiviral infection in the same three glioma cell lines. f Western blot showing SOX4 and FHL3 protein levels in T98G and U251 glioma cell lines overexpressing either FHL3 or SOX4 alone or co-overexpressing FHL3 and SOX4. g Growth curves in T98G and U251 glioma cells overexpressing either FHL3 or SOX4 alone or co-overexpressing both FHL3 and SOX4. Data are presented as the mean ± SD of three independent experiments. *P < 0.05

Article Snippet: Cell lines and cell culture The human glioma cell lines T98G, U87MG, and A172 were purchased from ATCC and cultured according to the guidelines recommended by the ATCC.

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Over Expression, Infection

Journal: Cell Death and Differentiation

Article Title: FHL3 links cell growth and self-renewal by modulating SOX4 in glioma

doi: 10.1038/s41418-018-0152-1

Figure Lengend Snippet: FHL3 suppresses SOX4 transcriptional activity and TGF-β-responsive transcription in a TGF-β1-independent manner. a Schematic diagram of the SOX4 promoter reporter construct. b, c T98G cells were co-transfected with an FHL3 overexpression construct (b) or FHL3 siRNAs (c), and either the dual luciferase reporter vector pEZX-PG04 or a SOX4 promoter reporter. Cells were treated with (+) or without (−) TGF-β1 and analyzed for Gaussia Luciferase (GLuc) and Secreted Alkaline Phosphatase (SEAP) activities, using the SEAP signal as an internal control. The normalized signals (ratio of GLuc to SEAP activities) are represented as the mean ± SD of three independent experiments. *P < 0.05 versus empty vector (b) or control siRNA (c) without TGF-β1. #P < 0.05 versus empty vector (b) or control siRNA (c) with TGF-β1. d T98G cells were co-transfected with the TGF-β signaling pathway reporter p3TP-Lux and either an FHL3 overexpression construct or empty vector. Cells were treated with (+) or without (–) TGF-β1 and analyzed for luciferase activity. Values are presented as the mean ± SD of three independent experiments. *P < 0.05 versus empty vector without TGF-β1. #P < 0.05 versus empty vector with TGF-β1. e Microarray results showing changes in the expression of TGF-β-responsive genes upon FHL3 overexpression. Red represents upregulated genes, while blue represents downregulated genes. f The correlation between FHL3 and SOX4 mRNA in TCGA GBM samples was analyzed on the LinkedOmics website using a Spearman correlation test. g Relative SOX4 and FHL3 protein levels in 13 grade II, 13 grade II–III, 7 grade III, 5 grade III–IV, and 16 grade IV glioma tissues compared with 7 normal brain tissue controls. Western blotting results were quantified using ImageJ software and are shown as the relative ratios of SOX4/β-Actin or FHL3/β-Actin protein levels (average values shown above the blots). *P < 0.05. P values were generated using an unpaired t-test

Article Snippet: Cell lines and cell culture The human glioma cell lines T98G, U87MG, and A172 were purchased from ATCC and cultured according to the guidelines recommended by the ATCC.

Techniques: Activity Assay, Construct, Transfection, Over Expression, Luciferase, Plasmid Preparation, Microarray, Expressing, Western Blot, Software, Generated

Journal: Data in Brief

Article Title: Polymerase chain reaction-based gene removal from plasmids

doi: 10.1016/j.dib.2015.04.024

Figure Lengend Snippet: Complete DpnI digestion can be achieved in Phusion master mix. (A)?The graphic representation of pCRII-U85 plasmid marked with four segments and 28 DpnI restriction sites. Purple: u85 , cyan: f1 , green: kanR , and yellow: ampR . The expected DpnI digestion pattern is shown on the right. Both the graphic representation and the digestion pattern were generated by a software named (A Plasmid Editor (APE) developed by M. Wayne Davis), (B)?DpnI digestion of the pCRII-U85 plasmid. A total amount of 400 ng plasmid was used in each reactions. All reactions were performed at 37 °C. Lane 1: DNA ladder, Lane 2: undigested DNA, Lane 3: 30 min digestion in the fast-digestion buffer from the vendor, Lane 4: 30 min digestion in 1× Pfusion Master Mix, Lane 5: 30 min digestion in water, Lane 6: 30 min digestion in 2× Pfusion Master Mix, Lane 6: 60 min digestion in 2× Pfusion Master Mix. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Phusion DNA polymerase master mix was purchased from NEB (Cat. #: M0531S).

Techniques: Plasmid Preparation, Generated, Software