Journal: Scientific reports

Article Title: TNFα-signal and cAMP-mediated signals oppositely regulate melanoma- associated ganglioside GD3 synthase gene in human melanocytes.

doi: 10.1038/s41598-019-51333-3

Figure Lengend Snippet: Figure 1. Synthetic pathway of sialic acid - containing glycosphingolipids, gangliosides and gene expression of glycosyltransferases in melanocytes and melanoma cell lines. (a) Glycosyltransferases genes for the synthesis of gangliosides are shown in italics. (b) Gene expression levels of glycosyltransferases in 2 melanocyte cells and 7 melanoma cell lines were analyzed. mRNA expressions of GD3 synthase, GM2/GD2 synthase, and GM1/GD1b synthase were analyzed by qRT-PCR in melanocytes (HEMn-LP (LP) and HEMn-MP (MP)) and melanoma lines (SK-MEL-28, SK-MEL-37, MeWo, SK-MEL-23, SK-MEL-31, SK-MEL-173, and SK-MEL-130). mRNA expression levels of these glycosyltransferases were normalized by those of human β-actin gene. Data represent means ± standard deviation (s.d.) (n = 4). Statistical analysis was performed by the two-tailed Student’s t-test (*P < 0.05; **P < 0.01).

Article Snippet: Antibodies used for detection of glycosyltransferases were as follows, GD3 Synthase (B-11) (a mouse monoclonal antibody) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA); GM2/GD2 Synthase (C-5) (a mouse monoclonal antibody) (Santa Cruz Biotechnology, Inc.); B3GALT4 Figure 8.

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, Standard Deviation, Two Tailed Test

Journal: Scientific reports

Article Title: TNFα-signal and cAMP-mediated signals oppositely regulate melanoma- associated ganglioside GD3 synthase gene in human melanocytes.

doi: 10.1038/s41598-019-51333-3

Figure Lengend Snippet: Figure 2. GD3 synthase gene upregulated by TNFα treatment or removal of dcAMP and IBMX from culture medium in melanocytes. (a) Time-course of mRNA expression levels of GD3 synthase, GM2/GD2 synthase, and GM1/GD1b synthase in melanocytes (LP) after adding TNFα to F10-A medium. Melanocytes were treated with TNFα (10 ng/mL) for 0–24 h. Then, mRNA expression levels were analyzed by qRT-PCR. mRNA expression levels of these glycosyltransferases were normalized by those of human GAPDH gene. Data represent means ± s.d. (n = 4). (b) Time-course of mRNA expression levels of glycosyltransferases in melanocytes (LP) after removing dcAMP (1 mM) and IBMX (0.1 mM) from F10-A medium. Melanocytes were cultured without dcAMP and IBMX for 0–24 h. Then, mRNA expression levels were analyzed by qRT-PCR. mRNA expression levels of these glycosyltransferases were normalized by those of human GAPDH gene. Data represent means ± s.d. (n = 4). Statistical analysis was performed by the two-tailed Student’s t-test (*P < 0.05; **P < 0.01). ns = not significant. (c) Immunofluorescence staining of GD3 of melanocytes (LP). Results on immunocytostaining with anti-GD3 antibody (R24) and Alexa Fluor 594-anti-mouse IgG were shown. Stained

Article Snippet: Antibodies used for detection of glycosyltransferases were as follows, GD3 Synthase (B-11) (a mouse monoclonal antibody) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA); GM2/GD2 Synthase (C-5) (a mouse monoclonal antibody) (Santa Cruz Biotechnology, Inc.); B3GALT4 Figure 8.

Techniques: Expressing, Quantitative RT-PCR, Cell Culture, Two Tailed Test, Immunofluorescence, Staining

Journal: Scientific reports

Article Title: TNFα-signal and cAMP-mediated signals oppositely regulate melanoma- associated ganglioside GD3 synthase gene in human melanocytes.

doi: 10.1038/s41598-019-51333-3

Figure Lengend Snippet: Figure 3. cAMP signal suppresses expression of GD3 synthase gene in melanocytes. (a) Melanocytes (LP) were cultured according to protocols 1~3. 1. Melanocytes were cultured in F10-A medium. 2. F10-A medium was exchanged with F10-A medium without dcAMP and IBMX (F10 medium) on day 5. 3. F10-A medium was exchanged with F10 medium on day 5, then dcAMP (1 mM) was added on day 6 and cultured for 12 h. (b) Melanocytes were cultured as shown in (a), and mRNA expression levels of GD3 synthase, GM2/GD2 synthase, and GM1/GD1b synthase were analyzed by qRT-PCR. mRNA expression levels of these glycosyltransferases were normalized by those of human GAPDH gene. Data represent means ± s.d. (n = 4). (c) Melanocytes (LP) were cultured according to protocols 1~4. 1. Melanocytes were cultured in F10-A medium. 2. F10-A medium was exchanged with F10 medium on day 5. 3. F10-A medium was exchanged with F10 medium on day 5, and dcAMP (1 mM) was added on day 6. 4. F10-A medium was exchanged with F10 medium on day 5. On day 6, α-MSH (100 nM) was added for 30 min, then medium was exchanged with F10 medium again. (d) Inhibitory effects of α-MSH on expression of the GD3 synthase gene in melanocytes. Melanocytes were cultured as shown in (c), and mRNA expression levels of GD3 synthase, GM2/GD2 synthase, and GM1/GD1b synthase were analyzed by qRT-PCR. mRNA expression levels of these glycosyltransferases were normalized by those of human GAPDH gene. Data represent means ± s.d. (n = 4). (e) Enhancing effects of a PKA inhibitor, H89, on expression of the GD3 synthase gene in melanocytes (LP). Melanocytes were cultured in F10-A medium

Article Snippet: Antibodies used for detection of glycosyltransferases were as follows, GD3 Synthase (B-11) (a mouse monoclonal antibody) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA); GM2/GD2 Synthase (C-5) (a mouse monoclonal antibody) (Santa Cruz Biotechnology, Inc.); B3GALT4 Figure 8.

Techniques: Expressing, Cell Culture, Quantitative RT-PCR

Journal: Scientific reports

Article Title: TNFα-signal and cAMP-mediated signals oppositely regulate melanoma- associated ganglioside GD3 synthase gene in human melanocytes.

doi: 10.1038/s41598-019-51333-3

Figure Lengend Snippet: Figure 4. Combined treatment of melanocytes with TNFα and H89 or TNFα and removal of dcAMP further enhances expression of the GD3 synthase gene. (a) Melanocytes (LP) were cultured according to protocols 1~6. Melanocytes were cultured in F10-A medium for 3 days. Then, 1. melanocytes were cultured in F10-A medium for 24 h, 2. cultured in F10-A medium without dcAMP and IBMX (F10 medium) for 24 h, 3. treated with TNFα (10 ng/mL) for 24 h, 4. treated with H89 (10 μM) for 24 h, 5. Melanocytes were cultured in F10 medium with TNFα (10 ng/mL) for 24 h, and 6. cultured with TNFα (10 ng/mL) and H89 (10 μM) for 24 h. (b) Melanocytes were cultured according to protocols 1~6 as in (a). mRNA expression levels of GD3 synthase, GM2/GD2 synthase, and GM1/GD1b synthase were analyzed by qRT-PCR. mRNA expression levels of these glycosyltransferases were normalized by those of human GAPDH gene. Data represent means ± s.d. (n = 4). Statistical analysis was performed by the two-tailed Student’s t-test (**P < 0.01).

Article Snippet: Antibodies used for detection of glycosyltransferases were as follows, GD3 Synthase (B-11) (a mouse monoclonal antibody) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA); GM2/GD2 Synthase (C-5) (a mouse monoclonal antibody) (Santa Cruz Biotechnology, Inc.); B3GALT4 Figure 8.

Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Two Tailed Test

Journal: Scientific reports

Article Title: TNFα-signal and cAMP-mediated signals oppositely regulate melanoma- associated ganglioside GD3 synthase gene in human melanocytes.

doi: 10.1038/s41598-019-51333-3

Figure Lengend Snippet: Figure 5. Inhibition of IKK results in suppression of GD3 synthase gene expression induced by TNFα in melanocytes. (a) Melanocytes (LP) were cultured according to protocols 1~4. Melanocytes were cultured in F10-A medium for 4 days. Then, 1. melanocytes were cultured in F10-A medium for 24 h, 2. cultured with TNFα (10 ng/mL) for 24 h, 3. cultured with TNFα (10 ng/mL) and an IKK inhibitor, WDL (12.5 μM or 25 μM), for 24 h, and 4. cultured with WDL (25 μM). (b) After treatment of melanocytes with or without TNFα and WDL as in (a), mRNA expression levels of GD3 synthase, GM2/GD2 synthase, and GM1/GD1b synthase were analyzed by qRT-PCR. mRNA expression levels of these glycosyltransferases were normalized by those of human GAPDH gene. Data represent means ± s.d. (n = 4). Statistical analysis was performed by the two-tailed Student’s t-test (*P < 0.05; **P < 0.01). ns = not significant.

Article Snippet: Antibodies used for detection of glycosyltransferases were as follows, GD3 Synthase (B-11) (a mouse monoclonal antibody) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA); GM2/GD2 Synthase (C-5) (a mouse monoclonal antibody) (Santa Cruz Biotechnology, Inc.); B3GALT4 Figure 8.

Techniques: Inhibition, Gene Expression, Cell Culture, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: Scientific reports

Article Title: TNFα-signal and cAMP-mediated signals oppositely regulate melanoma- associated ganglioside GD3 synthase gene in human melanocytes.

doi: 10.1038/s41598-019-51333-3

Figure Lengend Snippet: Figure 6. dcAMP, TNFα, and H89 do not affect expression levels of glycosyltransferases, while WDL suppresses GD3 synthase gene expression in melanomas. Melanoma cell lines, SK-MEL-28, SK-MEL-37, and MeWo were treated with or without TNFα (10 ng/mL) (a), dcAMP (1 mM) (b), and H89 (10 μM) (c) for 24 h. Then, mRNA expression levels of GD3 synthase, GM2/GD2 synthase, and GM1/GD1b synthase were analyzed by qRT-PCR. mRNA expression levels of these glycosyltransferases were normalized by those of human GAPDH gene. Data represent means ± s.d. (n = 4). (d) Treatment of melanoma cell lines (SK- MEL-28, SK-MEL-23, MeWo, and SK-MEL-130) with WDL reduced expression levels of the GD3 synthase gene. After treatment with or without WDL (20 μM) for 24 h, mRNA expressions levels of GD3 synthase, GM2/GD2 synthase, and GM1/GD1b synthase were analyzed by qRT-PCR. mRNA expression levels of these glycosyltransferases were normalized by those of human GAPDH gene. Data represent means ± s.d. (n = 3–4). Statistical analysis was performed by the two-tailed Student’s t-test (*P < 0.05; **P < 0.01). ns = not significant.

Article Snippet: Antibodies used for detection of glycosyltransferases were as follows, GD3 Synthase (B-11) (a mouse monoclonal antibody) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA); GM2/GD2 Synthase (C-5) (a mouse monoclonal antibody) (Santa Cruz Biotechnology, Inc.); B3GALT4 Figure 8.

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Two Tailed Test

Journal: Scientific reports

Article Title: TNFα-signal and cAMP-mediated signals oppositely regulate melanoma- associated ganglioside GD3 synthase gene in human melanocytes.

doi: 10.1038/s41598-019-51333-3

Figure Lengend Snippet: Figure 8. TNFα and cAMP-PKA signal pathways exert opposite effects to regulate GD3 synthase gene expression in human melanocytes. The TNFα signal pathway upregulated expression of the GD3 synthase gene, while cAMP-PKA signaling suppressed its expression.

Article Snippet: Antibodies used for detection of glycosyltransferases were as follows, GD3 Synthase (B-11) (a mouse monoclonal antibody) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA); GM2/GD2 Synthase (C-5) (a mouse monoclonal antibody) (Santa Cruz Biotechnology, Inc.); B3GALT4 Figure 8.

Techniques: Gene Expression, Expressing

Journal: Nature structural & molecular biology

Article Title: BRCA1 and S phase DNA repair pathways restrict LINE-1 retrotransposition in human cells.

doi: 10.1038/s41594-020-0374-z

Figure Lengend Snippet: Fig. 2 | Secondary validations of the DNA repair factors. a, Exon-junction qPCR analysis (green bars) and Arrayscan analysis (red bars) (n = 2 independent samples, error = s.d.). b, BRCA1 was knocked out in HCT116 cells. A BRCA1-expressing vector was transfected, and retrotransposition was quantified (% GFP+ cells). Recoded (ORFeus) or non-recoded (L1rp) L1 GFP-AI reporters were used. Expressed plasmids are indicated on the x axis; blue bars indicate wild-type HCT116 cells; red bars indicate BRCA1-deficient HCT116 cells (n = 2 independent samples, error = s.d.). c, Factors from the three major DSB repair pathways (HR, NHEJ, MMEJ) grouped, with their effects on L1 retrotransposition shown in a bar plot. The red bars indicate HR core factors (n = 2 independent samples, error = s.d.). d, Canonical FA factors and FA-like factors plotted for their effect on L1 retrotransposition. The factors within each group were ordered by their fold change compared to siScramble control (n = 2 independent samples). e, Immunoblot of iPOND inputs and immunoprecipitations of replication forks from ORFeus- or L1rp-expressing HeLa-M2 cells. The blotted proteins are indicated to the left of each panel. Cell treatments: #1 = BrdU for 10 min before fixation and CLICK reaction; #2 = BrdU for 10 min before fixation but no CLICK reaction; #3 = BrdU for 10 min before washing and incubation for 1 h in medium with thymidine 10 µM before fixation and CLICK reaction. If two panels are presented, they represent different exposures of the membrane. Quantification of ORF1p bands after iPOND are reported. Right, immunoblot of the indicated proteins using a prefixation aliquot of cells used for iPOND and expressing ORFeus or L1rp. Uncropped blot images are presented in Supplementary Data 1.

Article Snippet: We used the mouse BRCA1 [D9] antibody (Santa Cruz cat. sc-6954) at 1:100 dilution and FLAG-M2 antibody (Sigma, cat. number F1804) at 1:1,000 dilution for staining.

Techniques: Expressing, Plasmid Preparation, Transfection, Control, Western Blot, Incubation, Membrane

Journal: Nature structural & molecular biology

Article Title: BRCA1 and S phase DNA repair pathways restrict LINE-1 retrotransposition in human cells.

doi: 10.1038/s41594-020-0374-z

Figure Lengend Snippet: Fig. 4 | Re-expression of wild-type BRCA1 represses L1 retrotransposition in UWB1.289 ovarian carcinoma cells. a, Scheme of the ‘all-in-one’ Renilla-ORFeus-luciferase-AI retrotransposition reporter constitutively expressing the rtTA transactivator. b, L1 retrotransposition measurements as ratio of firefly (FF) over Renilla (RR) signals from UWB1.289 BRCA1-null cells (UWB) and UWB1.289 cells stably expressing a functional BRCA1 (UWB + BRCA1) transfected with the plasmid depicted in a or with a plasmid expressing RR alone. Cell were treated with 0.125 or 0.250 µg/ml doxycycline for 48 h before measurement of FF and RR (n = 16 independent samples, two-tailed distribution homoscedastic t test, ***P ≤ 0.005, ****P ≤ 0.001). The median, interquartile range and 95% confidence intervals are represented within the density plots (violin plots).

Article Snippet: We used the mouse BRCA1 [D9] antibody (Santa Cruz cat. sc-6954) at 1:100 dilution and FLAG-M2 antibody (Sigma, cat. number F1804) at 1:1,000 dilution for staining.

Techniques: Expressing, Luciferase, Stable Transfection, Functional Assay, Transfection, Plasmid Preparation, Two Tailed Test

Journal: Nature structural & molecular biology

Article Title: BRCA1 and S phase DNA repair pathways restrict LINE-1 retrotransposition in human cells.

doi: 10.1038/s41594-020-0374-z

Figure Lengend Snippet: Fig. 5 | HR/FA pathways repress L1 endonuclease-dependent and independent retrotransposition through BRCA1-mediated DNA resection. a, Retrotransposition assays using an L1 wild-type BSD-AI reporter. Clones of HeLa cells after blasticidin selection are stained with crystal violet. The number of cells plated per well and the transfected siRNA are indicated. b, Same experiment shown in a but using an endonuclease-dead (EN–) L1. Schematic representation of each of the L1-BSD-AI reporter plasmids used are shown at the bottom of each corresponding panel. c, L1 retrotransposition measurements using a firefly-AI reporter also expressing Renilla for normalization (Fig. 4a, without the rtTA). HeLa-M2 cells, expressing wild-type or H230A lacking endonuclease activity (L1 EN–) L1 luciferase-AI reporters, were plated in 3-cm wells and induced with doxycycline for 5 d. Retrotransposition activity is reported as the ratio (FF/RR) × 1,000 (n = 4 independent samples, error = s.d., two-tailed distribution homoscedastic t test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, blue * = significantly decreased compared to siCtrl, red * = significantly increased compared to siCtrl). qPCR measurements are presented in Supplementary Fig. 6b. d, BRCA1 and CtIP knockdowns promote resection at a DNA break, whereas 53BP1 knockdown inhibits resection at a DNA break. Resection can still occur when BRCA1 and its antagonist 53BP1 are both absent. e, Single and double knockdown of the indicated proteins in HeLa-M2 cells plated in 6-well plates. Fold change of L1 retrotransposition was calculated by normalizing %GFP upon knockdown to %GFP of scrambled siRNA-control-treated cells (n = 2 independent samples, error = s.d.). f, Retrotransposition measurements upon knockdown of the same factors depleted in e but using a pCEP-puro ORFeus-luciferase-AI and Renilla reporters episomally maintained in HeLa-M2 cells plated in 96-well plates (n = 8 independent samples, error = s.d., two-tailed distribution homoscedastic t test, ***P ≤ 0.001, blue * = significantly decreased compared to siCtrl, red * = significantly increased compared to siCtrl).

Article Snippet: We used the mouse BRCA1 [D9] antibody (Santa Cruz cat. sc-6954) at 1:100 dilution and FLAG-M2 antibody (Sigma, cat. number F1804) at 1:1,000 dilution for staining.

Techniques: Clone Assay, Selection, Staining, Transfection, Expressing, Activity Assay, Luciferase, Two Tailed Test, Knockdown, Control

Journal: Nature structural & molecular biology

Article Title: BRCA1 and S phase DNA repair pathways restrict LINE-1 retrotransposition in human cells.

doi: 10.1038/s41594-020-0374-z

Figure Lengend Snippet: Fig. 7 | BRCA1 inhibits ORF2 translation in the cytoplasm. a, Immunoblot of ORF1 and ORF2-FLAG proteins from control HeLa cells (siCtrl) or cells treated with the indicated siRNAs and expressing L1 ORFeus. Doxycycline (Dox) is used to induce the expression of L1; tubulin was used as a loading control. b, Left panel, immunoblot of HeLa cells expressing recoded L1 and BRCA1 knocked out using the indicated amounts of CRSIPR–Cas9 lentivirus. An empty plasmid (–BRCA1) or a plasmid encoding BRCA1 (+BRCA1) was also transfected, and L1 expression was induced for 48 h. Right, quantification of ORF2p normalized to tubulin. c, Immunofluorescence of HeLa cells expressing recoded L1 and treated for 24 h with control siRNA (siScramble) or siRNA against BRCA1 (siBRCA1). Scale bar, 100 µm d, BRCA1 mRNA quantification in different phases of the cell cycle using sorted FUCCI cells (n = 3 independent samples, error = s.d., two-tailed distribution homoscedastic t test). e,f, Quantification (e) and imaging (f) of ORF2p in cells expressing L1 and showing low or high BRCA1 (n = 3 independent samples, error = s.d., two-tailed distribution homoscedastic t test) f, L1 with halo-tagged ORF2p detected with JF657 dye (green). BRCA1 protein (red) detected with antibody D9. Red arrows indicate cells with low BRCA1 expression; blue arrows indicate cells with higher BRCA1 expression. Scale bar, 50 µm. g, L1 mRNA bound to BRCA1 measured by BRCA1 immunoprecipitation and qPCR compared to a standard curve of known concentrations of L1 DNA (n = 3 independent samples, error = s.d.). h, Left, cell cycle stages showed in HeLa S.FUCCI cells. Right, stacked bar plots showing the percentages of cells starting to express ORF2p in different cell cycle stages for control (CTR)- or siBRCA1-treated cells. The total numbers of quantified cells are reported beside the bars. Uncropped blot images for a and b are shown in Supplementary Data 1.

Article Snippet: We used the mouse BRCA1 [D9] antibody (Santa Cruz cat. sc-6954) at 1:100 dilution and FLAG-M2 antibody (Sigma, cat. number F1804) at 1:1,000 dilution for staining.

Techniques: Western Blot, Control, Expressing, Plasmid Preparation, Transfection, Immunofluorescence, Two Tailed Test, Imaging, Immunoprecipitation

Journal: Oncotarget

Article Title: Upregulated WEE1 protects endothelial cells of colorectal cancer liver metastases

doi: 10.18632/oncotarget.15039

Figure Lengend Snippet: a . WEE1 (left) and CDK1 (right) transcript abundances in CLMECs compared to matched liver endothelial cells (LiECs) (n=3 each). The data are from RNAseq analysis which is provided in detail in . b . On the left, Western blot labelled with anti-WEE1 and anti-β-Actin antibodies for LiECs and CLMECs. On the right, quantification of the CLMEC WEE1 band intensity relative to β-Actin and LiEC (n=3 each). c . On the left, Western blot labelled with anti-WEE1 and anti-β-Actin antibodies for non-transfected (NT) CLMECs and CLMECs transfected with scrambled siRNA (Scr) or WEE1-targeted siRNA (siWEE1). On the right, quantification of the WEE1 band intensity for the siWEE1 group normalised to the Scr group (n=3 each). d . On the left, Western blot labelled with anti-pCDK1-Y15, anti-CDK1 and anti-β-Actin antibodies for non-transfected (NT) CLMECs and CLMECs transfected with scrambled siRNA (Scr) or WEE1-targeted siRNA (siWEE1). On the right, quantification of the pCDK1-Y15 band intensity divided by the CDK1 intensity (n=3 each). e . On the left, fluorescence images of CLMECs 48 hr after transfection with scrambled (Scr) or WEE1 siRNA (siWEE1). Fluorescence was from cell nuclei stained with Vybrant® Dye Cycle™ (green). Scale bars 400 μm. On the right, normalised quantification of the images from independent repeats (n=3, N=18).

Article Snippet: Samples were transferred to PVDF membranes and labelled overnight with primary antibody: mouse anti-human β-Actin (1:2000, Santa Cruz Biotechnology), mouse anti-human WEE1 B-11 (1:300, Santa Cruz Biotechnology), mouse anti-human CD31 (1:1000, Dako, Clone JCT0A), rabbit anti-human pCDK1-Y15 (1:1000, Cell Signaling Technology), mouse anti-human CDK1 (1:1000, Cell Signaling Technology).

Techniques: Western Blot, Transfection, Fluorescence, Staining