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

Journal: PLoS Genetics

Article Title: Transcription Factor Oct1 Is a Somatic and Cancer Stem Cell Determinant

doi: 10.1371/journal.pgen.1003048

Figure Lengend Snippet: A. IF images of cross-sections from grossly uninvolved colon margins of a male familial adenomatous polyposis patient. Frozen sections were stained with mouse anti-Oct1 antibodies (Millipore MAB5434) and co-stained with TO-PRO. Crypts are shown in cross-section. White dashed circle highlights a crypt. Arrows indicate cells staining strongly for Oct1. B. IF images of colon crypt sections from a normal male individual. Sections were stained with DAPI, and anti-Oct1 and anti-ALDH1a1 antibodies. Merged images are shown at right. White dashed lines highlight crypts. Examples of cells co-staining with Oct1 and ALDH1a1 are highlighted with yellow arrows. An example cell staining with ALDH1a1 only is highlighted with an asterisk. Inset at lower right-hand corner is a digital magnification of the central portion of the image. Sections were formalin-fixed and paraffin-embedded. C. Frozen mouse colon tissue sections were stained with DAPI, and anti-Lrig1 and anti-Oct1 antibodies. IF images of longitudinal sections are shown. White dashed lines highlight the crypt. D. IF images of mouse small intestine sections. Sections were stained with DAPI and anti-Oct1 antibodies. Merged images are shown at right. White dashed lines highlight a crypt. Inset at upper right-hand corner is a digital magnification of the central portion of the image. Sections were formalin-fixed and paraffin-embedded. E. IF images of cross-sectional duodenum sections from a normal C57BL/6 mouse. Frozen sections were stained with DAPI and anti-Oct1 and anti-Lgr5 antibodies. Merged images are shown at right. Examples of co-staining cells are highlighted with yellow arrows. White dashed lines highlights crypts. F. Longitudinal sections.

Article Snippet: For panels 1D and 2A, two mixed rabbit anti-Oct1 antibodies (Bethyl, A301-716A, A301-717A) were used.

Techniques: Staining

Journal: PLoS Genetics

Article Title: Transcription Factor Oct1 Is a Somatic and Cancer Stem Cell Determinant

doi: 10.1371/journal.pgen.1003048

Figure Lengend Snippet: A. IF images are shown. Frozen malignant breast carcinoma sections (non-familial stage IIIA node-positive infiltrating ductal carcinoma, ER + PR + HER2 − ) were stained with mouse anti-Oct1 antibodies (Millipore MAB5434) and rabbit anti-ALDH1 antibodies (Abcam ab52492). Arrow indicates example double-positive cell. Asterisks show examples of an Oct1 HI cell with low ALDH1 and ALDH1 HI with low Oct1. Detail is shown in images below. B. Western blot depicting Oct1 levels in a panel of malignant epithelial cells isolated from pleural effusions (human breast carcinoma lung metastases). GAPDH is shown as a loading control. C. Examples of the CD24/44 profile from pleural effusions with highest and lowest Oct1 protein levels. D. Correlation of Oct1 protein and CD24 LO CD44 HI stem content in 15 individual patient samples (7 ER + PR + HER2 − , 5 ER − PR − HER2 − , 3 ER − PR − HER2 + ) collected from 14 different patients. One patient had tumor cells collected twice 22 months apart. Samples were placed into Oct1 LO and Oct1 HI categories based on Oct1 Western blot signal intensity relative to GAPDH staining. Cells were tested for percentage CD24 LO CD44 HI stem cell content and plotted. P -value was calculated using the two-tailed student T-test. E. The same analysis for Oct1 mRNA as measured by qRT-PCR.

Article Snippet: For panels 1D and 2A, two mixed rabbit anti-Oct1 antibodies (Bethyl, A301-716A, A301-717A) were used.

Techniques: Staining, Western Blot, Isolation, Control, Two Tailed Test, Quantitative RT-PCR

Journal: PLoS Genetics

Article Title: Transcription Factor Oct1 Is a Somatic and Cancer Stem Cell Determinant

doi: 10.1371/journal.pgen.1003048

Figure Lengend Snippet: A. Aldefluor staining profile of A549 cells infected with a doxycycline-inducible lentiviral shRNA. MFI was 169.0 for scrambled and 91.2 for Oct1-specific shRNA. A549 cells were infected with control or Oct1-specific lentiviral particles (Santa Cruz), selected with puromycin, and subjected to analysis after 48 hr. B. Efficacy of the A549 knockdown as assessed by Western blotting using anti-Oct1 antibodies and an anti-GAPDH loading control. C. Oct1 Western blot of unsorted normal A549 cells cultured under normal conditions, and sorted Aldefluor HI and Aldefluor LO populations is shown. GAPDH is used as a loading control. D. The same sorted cells or unsorted cells were subjected to qRT-PCR to determine Oct1 mRNA levels. Levels are show relative to GAPDH. Error bars depict standard deviations. E. Oct1 was ectopically expressed in A549 cells using a retrovirus (pBabe-Oct1) or empty vector. The mixed population of cells was subjected to selection with puromycin, and ALDH activity determined. F. Western blot using anti-Oct1 antibodies of the same cells shown in (E). ß-actin is shown as a loading control. G. Alignment of the Aldh1a1 promoter regions in several example vertebrate species. The conserved perfect octamer sequence centered at approximately −55 bp relative to the transcription start site is highlighted. Alignments were generated using a Clustal W-based algorithm within the Vector NTI software package (Invitrogen). Positions of PCR primer pairs for ChIP amplification are also shown. H. Quantification of ChIP enrichment using A549 cells and specific antibodies directed against Oct1, Mta2 (a component of the NuRD complex) and Jmjd1a. The PCR primer pair spanned the human Aldh1a1 octamer site. ChIP enrichment was quantified relative to isotype control anti-C/EBPß antibodies and relative to a control region as described in the section. Values are the average of four independent experiments. Error bars represent standard deviations.

Article Snippet: For panels 1D and 2A, two mixed rabbit anti-Oct1 antibodies (Bethyl, A301-716A, A301-717A) were used.

Techniques: Staining, Infection, shRNA, Control, Knockdown, Western Blot, Cell Culture, Quantitative RT-PCR, Plasmid Preparation, Selection, Activity Assay, Sequencing, Generated, Software, Amplification

Journal: PLoS Genetics

Article Title: Transcription Factor Oct1 Is a Somatic and Cancer Stem Cell Determinant

doi: 10.1371/journal.pgen.1003048

Figure Lengend Snippet: A. SP assay of luciferase-positive A549 cells carrying an inducible scrambled or Oct1-specific shRNA . Cells were treated with doxycycline for 4 days. Side population assays were conducted as described in the section. B. Western blot showing Oct1 protein levels in the two cell lines from (A) with and without 4-day culture in doxycycline. ß-actin is shown as a loading control. C. Quantification of the changed in SP percentage from three independent trials. Error bars denote standard deviations. D. Alignment of Abcg2 first intron regions in human and mouse. The perfect octamer sequence in both species is highlighted. Human Abcg2 has two annotated transcription start sites, so the element is located at both +14 kb and −12.4 kb relative to the transcription start sites. E. Quantification of ChIP enrichment using A549 cells and specific antibodies directed against Oct1, Mta2 and Jmjd1a. The PCR primer pair spanned the human Oct1 binding site in Abcg2 . ChIP enrichment was quantified relative to isotype control anti-C/EBPß antibodies and relative to a control region as described in the section. Values are the average of four independent experiments. Error bars represent standard deviations.

Article Snippet: For panels 1D and 2A, two mixed rabbit anti-Oct1 antibodies (Bethyl, A301-716A, A301-717A) were used.

Techniques: Luciferase, shRNA, Western Blot, Control, Sequencing, Binding Assay

Journal: PLoS Genetics

Article Title: Transcription Factor Oct1 Is a Somatic and Cancer Stem Cell Determinant

doi: 10.1371/journal.pgen.1003048

Figure Lengend Snippet: A. Numbers of nude mice engrafted in the left flank with A549 cells expressing the same Oct1-specific shRNA as shown in . Scrambled shRNAs in the contralateral flank were used as controls. The fraction of mice successfully engrafted is shown in tabular format. For the Oct1-specific and control shRNA lines, calculation of TIC frequency is shown at bottom. B. Images of engrafted tumors from animals receiving 1×10 5 cells. Left flank: scrambled shRNA. Right flank: Oct1-specific shRNA. C. Similar to (A), except human Oct1 was over-expressed in luciferase-expressing A549 cells using retroviral gene transduction. D. 4 th inguinal mammary fat pads of nude mice were engrafted with the indicated number of luciferase-expressing MDA-MB-231 cells expressing scrambled or Oct1-specific shRNAs. The fraction of engrafted animals is shown in tabular format. E. Western blot showing efficacy of lentiviral knockdown. ß-actin is shown as a loading control. F. Images of engrafted tumors from animals receiving 2×10 5 cells. Ventral view is shown. Right side: scrambled shRNAs. Left side: Oct1-specific shRNAs. G. Similar to (D) except human Oct1 was overexpressed using retroviral gene transduction. H. Western blot showing degree of Oct1 overexpression in cells used in (G). ß-actin is shown as a loading control.

Article Snippet: For panels 1D and 2A, two mixed rabbit anti-Oct1 antibodies (Bethyl, A301-716A, A301-717A) were used.

Techniques: Expressing, shRNA, Control, Luciferase, Retroviral, Transduction, Western Blot, Knockdown, Over Expression

Journal: PLoS Genetics

Article Title: Transcription Factor Oct1 Is a Somatic and Cancer Stem Cell Determinant

doi: 10.1371/journal.pgen.1003048

Figure Lengend Snippet: A. Peripheral blood leukocyte B and T cell profiles from primary and secondary animals transplanted with Oct1 deficient and WT littermate control fetal liver (FL). Cells were stained with anti-Thy1.2 and anti-B220 antibodies to reveal T and B cells. Control Rag1 −/− and WT animals are shown at the top for comparison. B. B and T cell repopulation is shown as a percentage of total cells for 5 independent experiments. C. Flow cytometry plots showing degree of T cell reconstitution in peripheral blood leukocytes in four Rag1 −/− recipient animals. For each animal, WT (top panels) or littermate Oct1 deficient (bottom panels) fetal livers were mixed with Thy1.1 + /Thy1.2 + WT bone marrow (BM) and injected retro-orbitally. Peripheral blood repopulation was analyzed at 10 and 15 weeks. D. The same peripheral blood analysis as in (C) was performed using Ly5.1/2 instead of Thy1.1/2 as a congenic marker. A single mouse for each condition is shown. E. Averages of 4 WT and 4 Oct1 deficient competitive repopulations as performed in (D) are shown. Peripheral blood was analyzed at 5 and 15 weeks. Error bars depict +/− standard deviation. F. Bone marrow from the mice in (D) were gated on LSK and analyzed for Ly5 expression. G. Averages of the 4 WT and 4 Oct1 deficient competitive repopulations at the level of LSK bone marrow precursors.

Article Snippet: For panels 1D and 2A, two mixed rabbit anti-Oct1 antibodies (Bethyl, A301-716A, A301-717A) were used.

Techniques: Control, Staining, Comparison, Flow Cytometry, Injection, Marker, Standard Deviation, Expressing