Journal: PLoS ONE

Article Title: A Microfluidic Platform for High-Throughput Multiplexed Protein Quantitation

doi: 10.1371/journal.pone.0117744

Figure Lengend Snippet: a) microfluidic design: the device consists of two PDMS layers: flow (blue) and control (red). The chip is an array of eight rows by 48 columns for 384 unit cells. Each unit cell is composed of: two antibody chambers divided by a reaction chamber (1–2), 4 MITOMI buttons (3), a valve that segregates the unit cells (4), a valve that separates antibody and reaction chambers (5) and a valve for releasing pressure in the antibody chambers (6). b) The PDMS chip is aligned to an epoxy-functionalized slide onto which primary and secondary antibodies were spotted. c) Assay details: schematic of the unit cell and cross section of a button region: i) functionalization of the surface: BSA-biotin is flowed though the chip followed by neutravidin. Next, the buttons are closed and BSA-biotin flowed again to passivate all neutravidin molecules except for those located underneath the MITOMI buttons, ii) the biotinylated primary antibody is allowed to diffuse into the MITOMI detection chamber and is bound by neutravidin immobilizing it in the MITOMI detection regions, iii) the sample is flown through the device and antigens are captured by the surface immobilized antibodies, iv) finally, the fluorescently labeled secondary antibody is allowed to diffuse into the MITOMI area, binds to the antigen if present, and is trapped by MITOMI. The entire device is then quantitated using a DNA microarray scanner.

Article Snippet: Neutravidin biotin-binding protein and the biotinylated bovine serum albumin BSA were purchased from Thermo Scientific (29130 and 31000 respectively).

Techniques: Control, Labeling, Microarray

Journal: Nucleic acids research

Article Title: Sustained pigmentation causes DNA damage and invokes translesion polymerase Polκ for repair in melanocytes.

doi: 10.1093/nar/gkad704

Figure Lengend Snippet: Figure 1. Melanin synthesis causes H2AX f oci f ormation DNA strand breaks and abasic sites formation. ( A ) Immunofluorescence of B16 cells treated with PTU and tyrosine with phosphoryla ted H2AX antibod y. Nuclear DNA stained with DAPI (blue) and H2AX in (red). Experiment was performed with two biological replicates and a representati v e image is depicted. Scale bar 10 m. ( B ) Quantitation of mean fluorescence intensity per cell of H2AX from two biological replicates of pigmented day 7 PTU or tyrosine treated cells (shown in A). Data r epr esented as a box plot, horizontal line r epr esents mean and whiskers r epr esent SEM. Ordinary one-way ANOVA was perf ormed f or multiple comparisons. Adjusted P -v alue: * P -v alue < 0.05, *** P - value < 0.001, **** P -value < 0.0001. ( C ) (Top) Cell pellet of day 7 B16 mouse melanoma cells grown at low density (100 cells / cm 2 ). Cells were left untreated for control treated with tyrosinase inhibitor 200 M phenylthiourea (PTU) or 1mM tyrosinase substrate L-tyrosine (Tyr) for 7 days. Number of cells, mean ± SEM across three biological replicates is depicted below the image of the cell pellet. Numbers r epr esent mean ± SEM cell counts across biological triplicates. (Bottom) Number of abasic sites in the genomic DNA was estimated by an aldehyde specific conjugation of biotin and subsequent detection using streptavidin based detection. Using standards, abasic sites per 10 5 bp is estimated. Bars r epr esent mean ± SEM across duplicate biological experiments, each conducted in triplicates. Ordinary one-w ay ANOVA w as performed for multiple comparisons * P -value < 0.05, ** P -value < 0.01, *** P -value < 0.001. ( D ) Single cell electrophoresis followed by comet analysis of B16 cells undergoing varying le v els of pigmentation in the presence of PTU and tyrosine (alkaline comet assay). Experiment was carried out at mid phase (day 5) and late phase (day 7) of pigmentation. Mean tail moment distribution across each population of duplicate biological experiments with atleast 50 comets analyzed is depicted by a violin plot. Two-way ANOVA was performed. Adjusted P values; ns non-significant, * P -value < 0.05, ** P -value < 0.01, *** P -value < 0.001, **** P -value < 0.0001. ( E ) Neutral comet assay on B16 unpigmented (day 0) and pigmented (day 7) cells. Mean tail moment distribution across each population of duplicate biological experiments with atleast 50 comets analyzed is depicted by a violin plot. Student’s unpaired t-test was performed. P values ns non-significant. ( F ) Single cell electrophoresis followed by comet analysis of B16 cells untr eated, tr eated with DMSO for 24 h, melanin synthesis ( ex-cellulo L-tyrosine and tyrosinase added to cell media) for 24 h or cells treated with 1 mM dihydroxyindole (DHI) for 24 h (alkaline comet assay). Mean tail moment distribution across each population of duplicate biological experiments with atleast 50 comets analyzed is depicted by a violin plot. Ordinary one-way ANOVA was performed. Adjusted P values: * P -value < 0.05, **** P -value < 0.00001.

Article Snippet: Cell lines, reagents and media B16 cells were obtained from ATCC.

Techniques: Immunofluorescence, Staining, Quantitation Assay, Fluorescence, Control, Conjugation Assay, Electrophoresis, Alkaline Single Cell Gel Electrophoresis, Neutral Comet Assay

Journal: Nucleic acids research

Article Title: Sustained pigmentation causes DNA damage and invokes translesion polymerase Polκ for repair in melanocytes.

doi: 10.1093/nar/gkad704

Figure Lengend Snippet: Figure 4. CRISPR-based genetic ablation of tyrosinase prevents melanogenesis, reduces DNA breaks and curtails Pol induction. ( A ) Immunofluorescence images of B16 WT and B16 Tyrosinase mutant (Tyr fs118 ) cells that w ere allow ed to pigment for 7 days. Bright field images indicate the presence of melanin granules. Labelling using 5-ethynyl-2 ′ -deoxyuridine (EdU) (red) and H2AX antibody (green). Merged images show co-localization of H2AX with EdU (yellow). A zoomed in view of a single cell is shown as an inset. Scale bars r epr esent 10 m. ( B ) (Top) Cell pellets of B16 WT and Tyrosinase mutant (Tyr fs118 ) cells on days 5, 6 and 7 of pigmentation. (Bottom) Quantitation of average of the number of H2AX puncta that are single positi v e or double positi v e (colocalizing with EdU puncta) per cell across 50 cells in B16 WT and B16 Tyr fs118 cells is depicted as a violin plot across three biological replicates. The percent population of cells with indicated puncta is mentioned alongside. ( C ) Growth curve analysis of B16 WT and Tyr fs118 cells on days 0, 5, 6 and 7 of pigmentation. Each point r epr esents mean ± SEM across biological triplicates. Two-w ay ANOVA w as performed. Adjusted P values: * P -value < 0.05, *** P -value < 0.001. ( D ) B16 WT and Tyr fs118 cells on days 5 and 7 of pigmentation were subjected to single cell electrophoresis and alkaline comet assay was performed. Mean tail moment distribution across each population of duplicate biological experiments with at least 50 comets analyzed is depicted by a violin plot. Two-w ay ANOVA w as performed. Adjusted P values ns non-significant: **** P -value < 0.0001. ( E ) Western blot analysis of whole-cell lysates of B16 WT and B16 Tyr fs118 cells on day 5 and day 7 stages of pigmentation. Numbers below the blot correspond to day 5 GAPDH normalized expression of the indicated protein. Experiments were performed in biological duplicates with similar results.

Article Snippet: Cell lines, reagents and media B16 cells were obtained from ATCC.

Techniques: CRISPR, Immunofluorescence, Mutagenesis, Quantitation Assay, Electrophoresis, Alkaline Single Cell Gel Electrophoresis, Western Blot, Expressing

Journal: Nucleic acids research

Article Title: Sustained pigmentation causes DNA damage and invokes translesion polymerase Polκ for repair in melanocytes.

doi: 10.1093/nar/gkad704

Figure Lengend Snippet: Figure 5. Normal human epidermal melanocytes (NHEM) respond to pigmentation induced DNA breaks by elevating Pol . ( A ) NHEM cells wer e tr eated with 200 M PTU or 1mM tyrosine for 7 days for differential pigmentation. (Top) Cell pellet, (bottom) western blot analysis of cell lysates with POLK, HSC70, phosphorylated H2AX, total H2AX and beta actin antibodies. Numbers below the blot correspond to control normalized expression of the indi- cated protein. Experiments were performed in biological duplicates. ( B ) Immunofluorescence of NHEM treated with PTU or tyrosine with phosphorylated H2AX antibody. Nuclear DNA stained with DAPI (blue) and H2AX in (red). Experiments were performed with two biological replicates. Scale bar 10 m. ( C ) Quantitation of mean fluorescence intensity per cell of H2AX from two biological replicates of NHEM treated with PTU or tyrosine (shown in B). Ordinary one-way ANOVA was performed for multiple comparisons. Adjusted P values: * P -value < 0.05, **** P -value < 0.0001. ( D ) PTU and tyrosine treated NHEM cells were subjected to single cell electrophoresis and comet analysis (alkaline comet assay). Mean tail moment distribution across each population of duplicate biological experiments with atleast 50 comets analyzed is depicted by a violin plot. Ordinary one-w ay ANOVA w as per- formed. Adjusted P values: ** P -value < 0.001, **** P -value < 0.00001. ( E ) Heat map of expression (fold change) in mRNA le v els of top two translesion polymerases (that were enriched in B16 microarray) (top), and a panel of known DNA replication stress response genes by qRT-PCR analysis in NHEM (Contr ol, PTU or tyr osine trea ted). Da ta r epr esented as mean of triplicate biological e xperiments. ( F ) Western b lot analysis of NHEM treated with DMSO or 50 nM AZ20, a selecti v e inhibitor of ATR kinase, for 24 h. Numbers below the blot correspond to control normalized expression of the indicated protein wrt beta-actin. Experiments were performed in biolo gical duplicates. ( G ) mRN A le v els of Polk in unpigmented B16 cells mock transfected, or with either control DNA, melanin modified DNA (plasmid DNA was incubated with L-DOPA and tyrosinase and column purified after 24 h) (Mel + DNA), DNA mixed with pre-synthesized melanin and coulmn purified [DNA+(Mel)], in-vitro melanin synthesis ( ex-cellulo L -tyrosine and tyrosinase added to cell media) for 24 h or cells treated with 1 mM DHI (DHI) for 24 h. Bars r epr esent per cent mRNA le v els compared to contr ol acr oss biological triplicates. Ordinary one-w ay ANOVA w as performed. Adjusted P v alues: * P -v alue < 0.05. (Inset) Western blot analysis of B16 cells transfected with onl y DN A (Con DNA) or melanin-modified DNA (Mel + DNA) with Pol antibody normalized to HSC70. Experiments were performed in biological triplicates.

Article Snippet: Cell lines, reagents and media B16 cells were obtained from ATCC.

Techniques: Western Blot, Control, Expressing, Immunofluorescence, Staining, Quantitation Assay, Fluorescence, Electrophoresis, Alkaline Single Cell Gel Electrophoresis, Microarray, Quantitative RT-PCR, Transfection, Modification, Plasmid Preparation, Incubation, Purification, Synthesized, In Vitro

Journal: Nucleic acids research

Article Title: Sustained pigmentation causes DNA damage and invokes translesion polymerase Polκ for repair in melanocytes.

doi: 10.1093/nar/gkad704

Figure Lengend Snippet: Figure 6. Silencing of Pol during pigmentation pre v ents replication stress response despite elevated DNA damage. ( A ) Cell pellets of control non-targeting (shNT) and Pol silenced (shPol ) B16 cells on day 0 (left) and day 7 (right) of pigmentation. ( B ) Immunofluorescence analysis of day 7 pigmented shNT and shPol cells with phosphorylated H2AX antibody (puncta labelled in green) and the nucleus is counterstained with DAPI (blue). Scale bars represent 10 m. ( C ) Quantitation of mean fluorescence intensity of H2AX (shown in B) from two biological replicates of shNT and shPol cells across day 0, 5 and 7 of pigmentation induction. Two-way ANOVA was performed for multiple comparisons. Adjusted P values * P -value < 0.05 *** P -value < 0.0005 **** P -value < 0.00001 ns non-significant. ( D ) shNT and shPol expressing pigmented B16 cells were subjected to single cell electrophoresis and comet analysis (alkaline comet) on days 0, 5 and 7 of pigmentation induction. Mean tail moment distribution across each population of duplicate biological experiments with at least 50 comets analyzed is depicted by a violin plot. Two-w ay ANOVA w as performed for multiple comparisons. Adjusted P values, ns non-significant, * P -value < 0.05. ( E ) Growth curve analysis of shNT and shPol expressing B16 cells on days 0, 5, 6 and 7 of pigmentation. Each point r epr esents mean ± SEM across biological triplicates. Two-way ANOVA was performed. Adjusted P values: * P -value < 0.05, *** P -value < 0.001. ( F ) Western blot analysis of DNA repair and cell cycle related proteins in shNT and shPol cells. Numbers below represent tubulin normalized fold changes wrt shNT. Experiments were performed in biological duplicates. ( G ) shNT and shPol expressing B16 cells were injected inside the flank of C57 / BL6 mice and allowed to grow as tumors. The volume of the tumor was non-invasi v ely monitored and plotted over time of biological triplicates mean ± SEM. Two-w ay ANOVA w as perf ormed f or multiple comparisons. Adjusted P v alues: * P -v alue < 0.05. ( H ) Heat ma p of expression (fold change) in mRN A le v els of a panel of known DNA replication stress response genes by qRT-PCR analysis in shNT (day 0-unpigmented and day 7-pigmented) and shPol (day 0-unpigmented and day 7-pigmented) B16 cells. ( I ) Western blot images and analysis of p-RPA2 and total RPA2 in shNT and shPol B16 cells (day 0 unpigmented and day 7 pigmented). Numbers below r epr esent beta-actin normalized fold changes wrt shNT at day 0. Experiments were performed in biolo gical triplicates. ( J ) Anal ysis of melanoma samples from TCGA data for mRNA expression of POLK (high, low or not detected) segregated into bar plots and proportion of mutations were plotted on y-axis. ( K ) Survival plot of melanoma patients with low or high expression of POLK from TCGA data. Analysis from Human Protein Atlas database. P air ed t -test P value 0.017.

Article Snippet: Cell lines, reagents and media B16 cells were obtained from ATCC.

Techniques: Control, Immunofluorescence, Quantitation Assay, Fluorescence, Expressing, Electrophoresis, Western Blot, Injection, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Monocytes Differentiate to Immune Suppressive Precursors of Metastasis-Associated Macrophages in Mouse Models of Metastatic Breast Cancer

doi: 10.3389/fimmu.2017.02004

Figure Lengend Snippet: Metastasis-associated macrophage (MAM) precursor cells (MAMPCs) suppress cytotoxicity of CD8 + T cells through a reactive oxygen species (ROS)-mediated mechanism. (A,B) Effects of myeloid cells on the CD8 + T cell-induced tumor cell apoptosis. Splenic CD8 + T cells from normal C57BL/6 mice were cultured with anti-CD3/CD28 antibodies (effector; E) in the absence or presence of MAMPCs, MAMs, or resident macrophages (RMACs) from the metastatic lung of E0771-LG-injected mice (MAMPC/E, MAM/E, RMAC/E, respectively). The preincubated T cells were then isolated and cultured with E0771-LG cells expressing red fluorescent protein in the nuclei (target; T) at the indicated E:T ratio in the presence of green fluorogenic caspase-3 substrate. After 36 h, the number of apoptotic cancer cells indicated by red/green double positive nuclei was counted. (A) Representative images of cells cultured with the caspase-3 substrate for 36 h (E:T = 4:1). Scale bar; 50 µm, arrowhead; apoptotic cancer cell. (B) Number of apoptotic cancer cells cultured with preactivated CD8 + T cells ( n = 3, two independent experiments). Data are mean ± SEM, * P < 0.01 vs. E + T (4:1), # P < 0.01 vs. MAMPC/E + T. (C) Fold-change of genes encoding checkpoint T cell receptor ligands in MAMPCs and MAMs compared with classical monocytes (C-MOs) determined by microarray analyses in Figure (logFC > 1, FDR < 0.05). Data on expression values are presented as mean ± SEM. Note that the scale is exponential. (D) Mean fluorescence intensity of checkpoint T cell receptor ligands assessed by flow cytometry in C-MOs, MAMPCs, and MAMs ( n = 3, two independent experiments). Blood (for C-MOs) and lung digestion (for MAMPCs and MAMs) were prepared from E0771-LG-injected C57BL/6 mice at 14 days posttumor injection and stained with antibodies for indicated markers or isotype IgG. Data are mean ± SEM, * P < 0.01 vs. IM, # P < 0.01 vs. MAMPC. (E) Effects of checkpoint inhibitors on the suppressive activity of MAMPCs and MAMs ( n = 6, two independent experiments). CD8 + T cells were cultured with anti-CD3/CD28 antibodies and neutralizing antibodies for PD1 or CTLA4, or isotype IgG in the absence (effector; E) or presence of MAMPCs (MAMPC/E) or MAMs (MAM/E). Cytotoxicity of the precultured CD8 + T cells against E0771-LG cells at 4:1 E/T ratio were assessed as described above. Data are mean ± SEM that represent the ratio in number of apoptotic cancer cells relative to that induced by CD8 + T cells cultured with IgG in the absence of MAMPCs or MAMs (control). * P < 0.01 vs. control, # P < 0.01 vs. IgG. (F) Effects of inhibitors of nitric oxide or ROS production on the suppressive activity of MAMPCs and MAMs ( n = 6, two independent experiments). CD8 + T cells were cultured with anti-CD3/CD28 antibodies and L-NMMA, nor-NOHA, catalase and superoxide dismutase (SOD) (Cat/SOD), or vehicle (–) in the absence (E) or presence of MAMPCs (MAMPC/E) or MAMs (MAM/E). Cytotoxicity of the precultured CD8 + T cells against E0771-LG cells at 4:1 E/T ratio were assessed as described above. Data are mean ± SEM that represent the ratio of apoptotic cancer cells relative to that induced by CD8 + T cells cultured with PBS in the absence of MAMPCs or MAMs (control). * P < 0.01 vs. control, # P < 0.01 vs. PBS.

Article Snippet: We intraperitoneally injected d -luciferin in PBS (GoldBio, 1.5 mg/100 μL/20 g mouse) into anesthetized E0771-LG:Fl tumor-bearing mice.

Techniques: Cell Culture, Injection, Isolation, Expressing, Microarray, Fluorescence, Flow Cytometry, Staining, Activity Assay

Journal: Scientific reports

Article Title: Peretinoin, an acyclic retinoid, inhibits hepatocarcinogenesis by suppressing sphingosine kinase 1 expression in vitro and in vivo.

doi: 10.1038/s41598-017-17285-2

Figure Lengend Snippet: Figure 1. Liver mRNA levels of SPHK1 in the PDGFC-Tg and Ath-HF diet mouse models. (a) mRNA level of SPHK1 in the liver of PDGF-C Tg mice determined by microarray analysis. We normalized the mRNA level of SPHK1 in the liver of PDGF-C Tg mice treated with or without 0.06% peretinoin to that of non-Tg mice. The mRNA levels were calculated from the microarray data published in one of our previous reports16. This figure shows the relative mRNA level of SPHK1 under each condition to that of non-Tg mice. Error bars indicate the standard deviation from three mice. The statistical significance of the difference in the average between the two groups was analyzed by the Student’s t test. (b) mRNA levels in the liver in the Ath-HF diet mouse model determined by microarray analysis. We normalized the mRNA level of SPHK1 in the liver of Ath-HF diet mice with or without 0.03% peretinoin to that of LF mice. The mRNA levels were calculated from the microarray data published in one of our previous reports17. This figure shows the relative mRNA level of SPHK1 under each condition to that in LF mice. Error bars indicate the standard deviation from three mice. The statistical significance was analyzed as above. (c) mRNA level of the liver in the Ath-HF diet mouse model determined by qRT-PCR. We quantitated the mRNA levels of SPHK1 and β-actin in the liver of Ath-HF diet mice with or without 0.03% peretinoin to that of LF mice by qRT-PCR (TaqMan assay); the mRNA level of SPHK1 was then normalized to that of β-actin for each mouse. The normalized mRNA levels of SPHK1 in the liver of Ath-HF diet mice with or without peretinoin were further normalized to that of LF mice. This figure shows the relative mRNA level of SPHK1 of each condition to that of LF mice. Error bars indicate the standard deviation from at least 10 mice. Statistical significance was analyzed as above. *p < 0.05, ***p < 0.005.

Article Snippet: The membranes were blocked in Blocking One or Blocking One-P solution (Nacalai Tesque, Kyoto, Japan), and the expression of SPHK1 was evaluated with rabbit anti-SPHK1 (#A302-177A; Bethyl Laboratories, Montgomery, TX), rabbit anti-Sp1 (#5931; Cell Signaling Technology, Danvers, MA), and rabbit β-actin antibody (#4967; Cell Signaling Technology).

Techniques: Microarray, Standard Deviation, Quantitative RT-PCR, TaqMan Assay

Journal: Scientific reports

Article Title: Peretinoin, an acyclic retinoid, inhibits hepatocarcinogenesis by suppressing sphingosine kinase 1 expression in vitro and in vivo.

doi: 10.1038/s41598-017-17285-2

Figure Lengend Snippet: Figure 2. mRNA level of SPHK1 in human liver. A. comparison of the mRNA level of SPHK1 between mild and severe fibrosis in the HCV-infected liver. (a) We performed liver biopsies on patients infected with HCV prior to antiviral treatment and quantified the mRNA levels of SPHK1 and β-actin in total RNA extracted from the liver by qRT-PCR (TaqMan assay); the mRNA level of SPHK1 was then normalized to that of β-actin for each patient. The patients were categorized into two groups based on their liver fibrosis grade; mild fibrosis was defined as METAVIR Score F1 or F2 and severe fibrosis as F3 or F4. We compared the normalized mRNA level of SPHK1 between mild and severe fibrotic liver. Error bars show the standard deviation from 119 patients with mild liver fibrosis and 59 patients with severe fibrosis. The statistical significance of the difference in the average between the two groups was analyzed by the Student’s t test. (b) Comparison of the mRNA level of SPHK1 before and after HCV eradication. We performed liver biopsies on another 12 patients before they started antiviral treatment for HCV and after successful eradication of HCV. We quantitated the mRNA levels of SPHK1 and β-actin in total RNA extracted from the liver by qRT-PCR (TaqMan assay); the mRNA level of SPHK1 was then normalized to that of β-actin for each patient. The relative mRNA level of SPHK1 after HCV eradication (post-SVR [sustained virologic response]) was normalized to that before antiviral treatment (prior to antiviral treatment) in the individual patients, with the mRNA level of SPHK1 before antiviral treatment set to 1. The statistical significance of the difference in the average between these two groups was analyzed by a paired t test. *p < 0.05, ***p < 0.005.

Article Snippet: The membranes were blocked in Blocking One or Blocking One-P solution (Nacalai Tesque, Kyoto, Japan), and the expression of SPHK1 was evaluated with rabbit anti-SPHK1 (#A302-177A; Bethyl Laboratories, Montgomery, TX), rabbit anti-Sp1 (#5931; Cell Signaling Technology, Danvers, MA), and rabbit β-actin antibody (#4967; Cell Signaling Technology).

Techniques: Comparison, Infection, Quantitative RT-PCR, TaqMan Assay, Standard Deviation

Journal: Scientific reports

Article Title: Peretinoin, an acyclic retinoid, inhibits hepatocarcinogenesis by suppressing sphingosine kinase 1 expression in vitro and in vivo.

doi: 10.1038/s41598-017-17285-2

Figure Lengend Snippet: Figure 3. Effects of peretinoin on the mRNA level, protein expression, and enzymatic activity of SPHK1. (a) effects of peretinoin on the mRNA level of sphingolipid-related genes in Huh-7 cells. Huh-7 cells were treated with peretinoin at 10, 25, or 50 μM or with 0.5% DMSO for 72 h and total cellular RNA was extracted. The mRNA levels of SPHK1, SPHK2, S1P lyase, S1P receptor 1–3 (S1P1, S1P2, and S1P3), and β-actin were quantitated by qRT-PCR (SYBR green assay), and the mRNA levels of SPHK1, SPHK2, S1P lyase, S1P1, S1P2, and S1P3 were normalized to that of β-actin. The relative mRNA level of each gene under each condition was normalized to that of DMSO control. (b) Effects of peretinoin on the protein expression of SPHK1 in Huh-7 cells. Huh-7 cells were treated with 0.5% DMSO or peretinoin 10, 20, or 40 μM. Total cell lysates were collected 12, 24, 48, and 72 h later and then probed by western blotting with anti-SPHK1 and β-actin antibodies. Full- length gels and blots before cropping are shown in supplemental Fig. S8. (c) Effects of peretinoin on the enzymatic activity of SPHK1 in Huh-7 cells. Huh-7 cells were treated with 0.5% DMSO or peretinoin at 10, 20, and 40 μM and a plasmid encoding SPHK1 cDNA or an empty vector were transfected into Huh-7 cells.

Article Snippet: The membranes were blocked in Blocking One or Blocking One-P solution (Nacalai Tesque, Kyoto, Japan), and the expression of SPHK1 was evaluated with rabbit anti-SPHK1 (#A302-177A; Bethyl Laboratories, Montgomery, TX), rabbit anti-Sp1 (#5931; Cell Signaling Technology, Danvers, MA), and rabbit β-actin antibody (#4967; Cell Signaling Technology).

Techniques: Expressing, Activity Assay, Quantitative RT-PCR, SYBR Green Assay, Control, Western Blot, Plasmid Preparation, Transfection

Journal: Scientific reports

Article Title: Peretinoin, an acyclic retinoid, inhibits hepatocarcinogenesis by suppressing sphingosine kinase 1 expression in vitro and in vivo.

doi: 10.1038/s41598-017-17285-2

Figure Lengend Snippet: Figure 4. Effects of peretinoin on SPHK1 promoter activity. A effects of peretinoin on the promoter activity of SPHK1 and SPHK2. (a) Plasmids encoding luciferase under the control of the promoter region of SPHK1, SPHK2, β-actin, and GAPDH were transfected into Huh-7 cells and, 24 h later, DMSO or peretinoin at 1, 5, 10, 20, or 40 μM was added. After 48-h DMSO or peretinoin treatment, luciferase activity was determined. The luciferase activity in each condition was normalized to that of DMSO control. Error bars indicate the standard deviation from three experiments and the statistical significance of the difference in the average was analyzed by one-way ANOVA. (b) Possible binding sites of Sp1 in the SPHK1 promoter region and a representation of deletion mutants. This figure shows the possible binding sites of Sp1 in the promoter region of SPHK1 and the series of deletion mutants analyzed. (c) Enhancement of the promoter activity of SPHK1 by Sp1 overexpression. A plasmid encoding Sp1 or a control empty vector was co-transfected with a plasmid encoding luciferase under the control of the SPHK1 promoter region and, 48 h later, luciferase activity was determined. Error bars indicate the standard deviation from three experiments and the statistical significance of the difference in the average was analyzed by the Student’s t test. (d) Restoration of the suppressive effects of peretinoin on the

Article Snippet: The membranes were blocked in Blocking One or Blocking One-P solution (Nacalai Tesque, Kyoto, Japan), and the expression of SPHK1 was evaluated with rabbit anti-SPHK1 (#A302-177A; Bethyl Laboratories, Montgomery, TX), rabbit anti-Sp1 (#5931; Cell Signaling Technology, Danvers, MA), and rabbit β-actin antibody (#4967; Cell Signaling Technology).

Techniques: Activity Assay, Luciferase, Control, Transfection, Standard Deviation, Binding Assay, Over Expression, Plasmid Preparation

Journal: Scientific reports

Article Title: Peretinoin, an acyclic retinoid, inhibits hepatocarcinogenesis by suppressing sphingosine kinase 1 expression in vitro and in vivo.

doi: 10.1038/s41598-017-17285-2

Figure Lengend Snippet: Figure 5. Effects of SPHK1 knockout on hepatocarcinogenesis in a DEN-induced hepatoma mouse model. DEN was injected into the peritoneal cavity of 2-week-old SPHK1 knockout, SPHK1 Tg, and wild-type mice at 25 mg/kg, and then those mice were sacrificed at 40 weeks old. (a) The numbers of mice that had a liver tumor at sacrifice. (b) The numbers of liver tumors in each mouse. (c) The maximum liver tumor size in individual mice that developed hepatoma. (d) Macrophotographs of representative mouse livers at sacrifice. (e) High power field microphotographs of representative mouse liver tumors stained with hematoxylin and eosin. Error bars shows the standard deviation from at least 20 mice, and the statistical significance of the difference in the average between these two groups was analyzed by the Student’s t test. *p < 0.05, ***p < 0.005

Article Snippet: The membranes were blocked in Blocking One or Blocking One-P solution (Nacalai Tesque, Kyoto, Japan), and the expression of SPHK1 was evaluated with rabbit anti-SPHK1 (#A302-177A; Bethyl Laboratories, Montgomery, TX), rabbit anti-Sp1 (#5931; Cell Signaling Technology, Danvers, MA), and rabbit β-actin antibody (#4967; Cell Signaling Technology).

Techniques: Knock-Out, Injection, Staining, Standard Deviation