Journal: Nature Communications

Article Title: ICAM1 initiates CTC cluster formation and trans-endothelial migration in lung metastasis of breast cancer

doi: 10.1038/s41467-021-25189-z

Figure Lengend Snippet: a A schematic showing the single-cell RNA sequencing of the sorted cells from L2T- or L2G-labeled TNBC PDX (mice 1 and 2) or MDA-MB-231 tumor (mouse 3)-bearing mice, both primary breast tumors and lung metastases (early micrometastases). b Heatmap denoting expression magnitude estimates in log scale (red color) for ICAM1 and co-expressed stemness signature genes in primary tumor cells and lung metastases ( N = 3 mice, 2 with TN PDXs and 1 with MDA-MB-231 tumor). Genes are sorted according to their correlation with ICAM1 across all cells, as denoted by the gray bars on the right (top highest to bottom lowest). The bottom list of CD44, GAPDH, ACTB, eGFP, and tdTomato serve as control genes without significant changes between primary tumor cells and lung metastases. c Representative ICAM1 expression in L2T + or L2G + primary tumor cells and lung metastases determined by flow cytometry from different breast cancer PDX models (TN1, TN2, and TN3). Flow profile gates are shown in Supplementary Fig. . d Quantitative data of the differential ICAM1 expression in PDX primary tumors versus lung metastasis from c . n = 3 biological replicates (mice) for each model. One-sided t test * P = 0.04; ** P = 0.01; * P = 0.02. The boxes range from the first to third quartile with x in a box indicating mean value and whisker lines extending to outliers (minimum and maximum). e Representative IHC staining images of ICAM1 expression (brown color) in primary tumors and lung metastases from PDX TN1 and TN2 models validating c and d . f Distribution of ICAM1 expression across PAM50 subtypes in the TCGA BRCA cohort ( N = 1037). Basal-like and HER2-enriched subtypes are the top two exhibiting significantly higher ICAM1 expression as compared to normal breast tissue (percentage of cases above the blue line value are shown for each subtype). Statistical significance was assessed using a two-sided Student’s t test. * P < 0.05, ** P < 0.01, and **** P ≤ 0.0001. g Schematic and flow histogram analyses of the orthotopically implanted TN1-PDX tumors with or without ICAM1 overexpression (OE) at the 4th mammary fat pads. h . PDX TN1 tumor weights 2 months after orthotopic injections of TN1 cells with ICAM1 OE and control vector (Con) (2.5e5 cells into one mammary fat pad/mouse). n = 3 mice per cell group. Two-sided t test * P = 0.04. The boxes range from the first to third quartile with x in a box indicating mean value and whisker lines extending to outliers (minimum and maximum). i , j Representative lung images and normalized BLI signals (total flux) of spontaneous lung metastases in orthotopically implanted ICAM1 OE and control TN1 tumor-bearing mice as in g , h . n = 3 mice per group. Two-sided t test ** P = 0.003. The boxes range from the first to third quartile with x in a box indicating mean value and whisker lines extending to outliers (minimum and maximum). k Schematic and flow histogram analyses of tail vein injected MDA-MB-231 tumor cells transfectd with siRNA control (siCon) and siICAM1. l , m . Representative images and quantitative data of BLI signal of mice injected with siCon and siICAM1-transfected MDA-MB-231 cells via tail vein ( n = 4 mice per cell group. Two-sided t test ** P = 0.01 for time point comparisons. N = 6 independent experiments). The boxes range from the first to third quartile with x in a box indicating mean value and whisker lines extending to outliers (minimum and maximum).

Article Snippet: For overexpression of ICAM1 in HEK-293 cells, the following plasmids were transfected into HEK-293 cells by PolyJet (SignaGen Laboratories): ICAM1 cDNA ORF Clone, Human, C-Flag tag (Sino Biological, Cat# HG10346-CF); and ICAM-1 cDNA ORF Clone, Human, C-Myc tag (Sino Biological, Cat# HG10346-CM).

Techniques: RNA Sequencing Assay, Labeling, Expressing, Flow Cytometry, Whisker Assay, Immunohistochemistry, Over Expression, Plasmid Preparation, Injection, Transfection

Journal: Nature Communications

Article Title: ICAM1 initiates CTC cluster formation and trans-endothelial migration in lung metastasis of breast cancer

doi: 10.1038/s41467-021-25189-z

Figure Lengend Snippet: a Representative IHC staining images of ICAM1 − single CTC and ICAM1 + CTC cluster (5-cell) (brown) in the lung vasculature of the PDX TN1-bearing mouse. Minimal 3 sets of independent images have shown similar patterns. b Representative images of CellSearch-analyzed CTCs in breast cancer (BC) patients: CD45 − , cytokeratin (CK) + (green), DAPI + (purple), ICAM1 − or ICAM1 + (gray) (two single cells and a three-cell cluster). c Bar graph of the proportion of flow analyzed CD45 − ICAM1 + single (blue) and clustered (orange) CTCs in each of 51 stage III–IV BC patients ( N = 51 patients. Two-sided t test **** P = 0.00007). d , e Representative images ( d ) and quantitative cluster counts ( e ) of ICAM1 + and ICAM1 − cells sorted from PDX TN1 OE models showing different cluster formation efficiencies ex vivo ( n = 10 biological replicates. Data are presented as mean values ± SEM. Error bars represent SE values. Two-sided t test * P < 0.02. N = 2 independent experiments). f , g Representative images ( f ) and quantified cluster sizes ( g ) of MDA-MB-231 cells transfected with siRNA control (Con) and siICAM1, and resuspended in poly-HEMA treated plates for cluster formation ( n = 10 biological replicates. Data are presented as mean values ± SD. Error bars represent SD values. Two-sided t test *** P = 0.0003. N = 4 independent experiments). h , i Diagram of solid phase self-interaction assay ( h ) and quantified binding ( i ) of biotin-conjugated ICAM1 and BSA at 1 μg to the solid phase coated with ICAM1 (1 µg), measured as OD 450 units (two-sided t test **** P = 0.0000003. N = 2 independent experiments). The boxes range from the first to third quartile with x in a box indicating mean value and whisker lines extending to outliers (minimum and maximum). j Co-IP detection of ICAM1-Flag and ICAM-Myc intercellular homodimers. Upper panel, diagram of two HEK-293T cells transfected with C-terminal Flag-tagged and Myc-tagged ICAM1, respectively. Lower panel, immunoblots for Flag, Myc, and ICAM1, respectively, following co-IP with anti-Flag antibody. N = 3 independent experiments. k ICAM1 extracellular homodimer structure model with interacting domains between II-IV, III-III, and IV-II of two molecules extended from opposite directions. l Co-IP detection of ICAM1-Flag with ICAM-Myc variants transfected in two sets of HEK293T cells separately. Upper panel, diagram of two HEK-293T cells transfected with ICAM1-Flag (WT) and ICAM1-Myc variants (1 of 5 different variants). Lower panel, immunoblots for Flag and Myc, following co-IP with anti-Flag showing lost interactions with mutant ICAM1. Colored asterisks indicate the various WT and variant constructs ( N = 2 independent experiments).

Article Snippet: For overexpression of ICAM1 in HEK-293 cells, the following plasmids were transfected into HEK-293 cells by PolyJet (SignaGen Laboratories): ICAM1 cDNA ORF Clone, Human, C-Flag tag (Sino Biological, Cat# HG10346-CF); and ICAM-1 cDNA ORF Clone, Human, C-Myc tag (Sino Biological, Cat# HG10346-CM).

Techniques: Immunohistochemistry, Ex Vivo, Transfection, Binding Assay, Whisker Assay, Co-Immunoprecipitation Assay, Western Blot, Mutagenesis, Variant Assay, Construct

Journal: Nature Communications

Article Title: ICAM1 initiates CTC cluster formation and trans-endothelial migration in lung metastasis of breast cancer

doi: 10.1038/s41467-021-25189-z

Figure Lengend Snippet: a Down-regulated pathways upon ICAM1 knockdown in MDA-MB-231 cells, analyzed by RNA sequencing (top) and mass spectrometry analysis (bottom). b GSEA of the gene sets for histone deacetylase targets, H3K27ME3, and EZH2 targets enriched among the up-regulated genes in MDA-MB-231 ICAM1 knockdown cells in comparison to siRNA control, identified by RNA sequencing. c Immunoblots of ICAM1 and CDK6 in MDA-MB-231 cells transfected with control siRNAs (siCon) and siICAM1 for gene knockdown. N = 3 independent experiments. d , e Representative images ( d ) and mammosphere quantitation ( e ) of MDA-MB-231 cells transfected with siCon, siICAM1, and siCDK6 ( n = 3 biological replicates, two-sided t test ** P = 0.006; *** P = 0.0008. N = 3 independent experiments. The boxes range from the first to third quartile with x in a box indicating mean value and whisker lines extending to outliers (minimum and maximum). f – h Representative images ( f ) and quantitative data ( g , h ) of BLI signals of mice injectd with siCon, siICAM1, and siCDK6-transfected MDA-MB-231 cells via tail vein ( n = 3 mice per cell group. g Data are presented as mean values ± SD, two-sided t test * P < 0.05; ** P ≤ 0.01. N = 4 independent experiments. h The boxes range from the first to third quartile with x in a box indicating mean value and whisker lines extending to outliers (minimum and maximum). i , j Representative images ( i ) and counts ( j ) of proliferative MDA-MB-231 (50k cells/well) transfected with siCon, siICAM1, and siCDK6. Cell numbers were measured via hemocytometer counting 48 h after seeding ( n = 4 biological replicates. two-sided t test * P = 0.04; ** P = 0.01. N = 3 independent experiments. The boxes range from the first to third quartile with x in a box indicating mean value and whisker lines extending to outliers (minimum and maximum). k Pearson’s pairwise correlation plot of ICAM1 versus CDK6 expression in breast cancer patients ( n = 4712), by BC Gene-Expression Miner v4.4. ICAM1 vs. CDK6 R = 0.40. **** P < 0.0001.

Article Snippet: For overexpression of ICAM1 in HEK-293 cells, the following plasmids were transfected into HEK-293 cells by PolyJet (SignaGen Laboratories): ICAM1 cDNA ORF Clone, Human, C-Flag tag (Sino Biological, Cat# HG10346-CF); and ICAM-1 cDNA ORF Clone, Human, C-Myc tag (Sino Biological, Cat# HG10346-CM).

Techniques: RNA Sequencing Assay, Mass Spectrometry, Histone Deacetylase Assay, Western Blot, Transfection, Quantitation Assay, Whisker Assay, Expressing

Journal: Nature Communications

Article Title: ICAM1 initiates CTC cluster formation and trans-endothelial migration in lung metastasis of breast cancer

doi: 10.1038/s41467-021-25189-z

Figure Lengend Snippet: a Diagram of TEM assay with HUVEC cell-coated transwell inserts and tumor cells seeded on the top. Tumor cells transmigrated to the bottom were measured 24 h after seeding. (MDA-MB-231 tumor cells; green, HUVEC; purple, and ICAM1 protein; blue). b Representative images (top) and quantitative analyses (bottom) of TNBC cells transmigrated to the bottom chamber, including four conditions (from left to right): (1) siRNA control tumor cells and HUVECs; (2) ICAM1 knockdown in endothelial cells (EC); (3) ICAM1 knockdown in tumor cells; (4) ICAM1 knockdown in both tumor cells and HUVECs ( n = 3 biological replicates. Two-sided t test * P = 0.04; ** P = 0.007; *** P = 0.0006. NS not significant. The boxes range from the first to third quartile with x in a box indicating mean value and whisker lines extending to outliers (minimum and maximum). c , d Time-lapse co-culture images at 0 and 24 h of incubation with TNBC cells (green) and endothelial cells (red) ( c ) and quantitative analyses of aggregates ( d ) following ICAM1 knockdown in both cell types ( n = 8 biological replicates. Data are presented as mean values ± SD. Error bars represent SD values. Two-sided t test **** P = 0.0000005). e , f Representative images ( e ) and quantitative analyses ( f ) of TNBC cell cluster formation in the presence of IgG control or anti-ICAM1 neutralizing antibody ( n = 10 biological replicates, Data are presented as mean values ± SEM. Error bars represent SE values. Two-sided t test ** P = 0.005). g , h Diagram and representative images of TEM assay ( g ) and quantitative analysis ( h ) of breast tumor cells transmigrated to the bottom chamber in the presence of IgG control or anti-ICAM1 neutralizing antibody ( n = 3 biological replicates. Two-sided t test ** P = 0.002). (Diagram: MDA-MB-231 tumor cells; green, HUVEC; purple, ICAM1 protein; blue, and anti-ICAM1 antibody; yellow). The boxes range from the first to third quartile with x in a box indicating mean value and whisker lines extending to outliers (minimum and maximum).

Article Snippet: For overexpression of ICAM1 in HEK-293 cells, the following plasmids were transfected into HEK-293 cells by PolyJet (SignaGen Laboratories): ICAM1 cDNA ORF Clone, Human, C-Flag tag (Sino Biological, Cat# HG10346-CF); and ICAM-1 cDNA ORF Clone, Human, C-Myc tag (Sino Biological, Cat# HG10346-CM).

Techniques: Whisker Assay, Co-Culture Assay, Incubation

Journal: Nature Communications

Article Title: ICAM1 initiates CTC cluster formation and trans-endothelial migration in lung metastasis of breast cancer

doi: 10.1038/s41467-021-25189-z

Figure Lengend Snippet: a Differences in distant metastasis-free survival (DMFS) of patients with breast tumors exhibiting high versus low ICAM1 expression in the GSE25055 ( N = 306) cohort. Note that patients with tumors expressing higher than median ICAM1 expression (red) are associated with significantly poorer DMFS (Logrank P = 0.035). b Differences in disease-specific survival (DSS) of patients with breast tumors exhibiting high versus low ICAM1 expression alone (left panel) or in combination with a 98-gene Stemness Signature Index (right panel) in the GSE1456-GPL96 ( N = 159) cohort. Note that patients with tumors harboring higher than median ICAM1 expression (left panel, red) exhibit significantly poorer DSS (Logrank P = 0.025). Similarly, breast cancers expressing higher than median Stemness Signature Index in addition to higher than median ICAM1 expression (Dual-High, red) exhibit poorer DSS (Logrank P = 0.034). c , d Representative images ( c ) and quantitative data ( d ) of BLI signals in mice on Day 0 (D0, 0 h) and Day 1 (D1, 24 h, dissected lungs) after injection of sorted ICAM1 + and ICAM1 − TN1 PDX cells via tail vein ( n = 4 mice per cell group. Two-sided t test ** P = 0.005). The boxes range from the first to third quartile with x in a box indicating mean value and whisker lines extending to outliers (minimum and maximum). e – g Representative BLI images of mice at 0 h and dissected lungs at 10 h after tumor cell injections via tail vein ( e ), normalized metastatic seeding to the lungs ( f ), and L2G + CTC analysis (%) in the blood ( g ). The mice were pretreated with IgG or anti-ICAM1 neutralizing antibody (80 µg/mouse) via tail vein, and 3 h later followed by a tail vein injection of MDA-MB-231 cells (1 × 10 5 cells) and the antibody (100 μg, pre-incubated with cells for 30 min). Lungs were collected 10 h after injection. ( n = 3 mice per group. Two-sided t test ** P = 0.008; ** P = 0.01. N = 2 independent experiments. The boxes range from the first to third quartile with x in a box indicating mean value and whisker lines extending to outliers (minimum and maximum). h , i Representative BLI images of mice on day 0 post orthotopic implantations of MDA-MB-231 tumor cells, pictures of dissected breast tumors, and BLI of dissected lungs ( h ) and quantitative lung metastasis after normalization by tumor weight of each mouse ( i ). The mice were given long-term treatment with IgG or anti-ICAM1 antibody (100 µg/mouse, twice a week for 4 weeks) ( n = 3 mice per group. Two-sided t test ** P = 0.01). The boxes range from the first to third quartile with x in a box indicating mean value and whisker lines extending to outliers (minimum and maximum). j Diagram of ICAM1 + tumor cells initiating multicellular cluster formation in the circulation, directing TEM, and mediating lung metastasis; partially through sustained expression of CDK6. Blocking the ICAM1 intercellular homophilic interactions between tumor-tumor and tumor-endothelial cells with neutralizing antibodies (gold) will inhibit CTC cluster formation and TEM, and eventually decrease or block metastasis. (ICAM1 + tumor cells; green, ICAM1 protein; blue, ICAM1 − tumor cells; beige, Endothelial cells; purple, and anti-ICAM1 antibody; yellow).

Article Snippet: For overexpression of ICAM1 in HEK-293 cells, the following plasmids were transfected into HEK-293 cells by PolyJet (SignaGen Laboratories): ICAM1 cDNA ORF Clone, Human, C-Flag tag (Sino Biological, Cat# HG10346-CF); and ICAM-1 cDNA ORF Clone, Human, C-Myc tag (Sino Biological, Cat# HG10346-CM).

Techniques: Expressing, Injection, Whisker Assay, Incubation, Blocking Assay

Journal: bioRxiv

Article Title: Arsenic induces two different interaction modes of SUMO with promyelocytic leukemia (PML) proteins

doi: 10.1101/2023.04.24.538205

Figure Lengend Snippet: (A) The cells were treated with 3 μM As 3+ (+) or left untreated (-) for 2 h. The soluble (Sol) and insoluble (Ins) fractions of cell lysates were obtained from three different wells. The membrane was probed sequentially with anti-SUMO2/3, anti-PML, and finally with HRP-tagged anti-tubulin and anti-histone H3 antibodies. Note that the polyclonal anti-PML antibody detected endogenous PML isoforms in HEK293 cells. PMLs in the soluble fraction were partially shifted to the insoluble fraction, and SUMOylated by 2-h exposure to 3 μM As 3+ in HEK293 cells. An open arrowhead indicates SUMO2/3 monomers. (B) The cells were treated with 3 μM As 3+ for 2 h or left untreated before fixation and immunostaining with anti-PML and anti-SUMO2/3 antibodies. Note that small PML dots co-localized with SUMO2/3 dots in the nuclei of HEK293 cells. Scale bar = 10 μm. (C) HEK293, HEKPML, and PML -/- cells were treated with 3 μM As 3+ or left untreated for 1 h. The cells were fixed, permeabilized, and stained with Alexa Fluoro 488-labeled anti-SUMO2/3 antibody. The nuclei were counterstained with DAPI. Note that SUMO2/3 was stained diffusely over the nuclei of PML -/- cells, and the nucleoplasmic PML (PML-VI) was more restricted to PML-NBs in As 3+ -exposed HEKPML cells. The size of PML-NBs (SUMO2/3 dots) was assayed by the estimated diameter assuming that the PML-NBs were spherical. Measurements were performed for more than 200 dots (201 – 212 dots) using ImageJ and presented as means ± SD. *, Both the number and the size of SUMO dots were significantly different between HEK293 and HEKPML cells. The size was slightly, but significantly different between untreated (-) and As 3+ -exposed (+) cells (p value = 0.018) as determined by two-way ANOVA.

Article Snippet: HEK293, which were stably transfected with the human PML-VI gene (RC220236, OriGene, Rockville, MD), were selected using neomycin and were designated as HEKPML cells [ ].

Techniques: Immunostaining, Staining, Labeling

Journal: bioRxiv

Article Title: Arsenic induces two different interaction modes of SUMO with promyelocytic leukemia (PML) proteins

doi: 10.1101/2023.04.24.538205

Figure Lengend Snippet: (A) N-terminal mCherry expression plasmids harboring wild, K11R mutant (K11R), and C-terminal GG-deleted (ΔGG) SUMO2 were stably transduced in HEK293 cells. The cells were treated with 3 μM As 3+ for 2 h (As (+)) or left untreated (As (-)). The soluble (Sol) and insoluble (Ins) fractions of each cell lysate were analyzed for SUMO1, SUMO2/3, PML, and RFP by western blotting. Closed and open triangles indicate mCherry-conjugated SUMO2 monomer and endogenous SUMO monomer, respectively. 293, non-transfected HEK293 cells. The right half parenthesis indicates SUMOylated proteins with mCherry-conjugated SUMO2.

Article Snippet: HEK293, which were stably transfected with the human PML-VI gene (RC220236, OriGene, Rockville, MD), were selected using neomycin and were designated as HEKPML cells [ ].

Techniques: Expressing, Mutagenesis, Stable Transfection, Western Blot, Transfection

Journal: bioRxiv

Article Title: Arsenic induces two different interaction modes of SUMO with promyelocytic leukemia (PML) proteins

doi: 10.1101/2023.04.24.538205

Figure Lengend Snippet: HEK293 cells stably expressing mCherry-conjugated SUMO2 (Wild) or mCherry-conjugated SUMO2 with C-terminal diglycine deletion (ΔGG) were used. The cells were treated with 3 μM As 3+ (As) or left untreated (C) for 1 h and lyzed with RIPA buffer. (A) The cell lysates were separated into the soluble (Sol) and insoluble fractions (Ins) and analyzed for PML, SUMO1, SUMO2/3, and mCherry-conjugated SUMO2 (RFP) by western blotting. (B) The clear supernatants of the lysates were used for immunoprecipitation assay using anti-RFP-conjugated magnetic beads. Note that PML and SUMOylated PML were pulled down by ΔGG mCherry-SUMO2, suggesting that PML and PML SUMOylated with endogenous SUMO molecules associated with SUMO2 non-covalently in As 3+ -exposed HEK293 cells. An open triangle indicates endogenous SUMO1 or SUMO2/3 monomers. Closed and open triangles indicate mCherry-conjugated SUMO2 monomer and endogenous SUMO monomer, respectively.

Article Snippet: HEK293, which were stably transfected with the human PML-VI gene (RC220236, OriGene, Rockville, MD), were selected using neomycin and were designated as HEKPML cells [ ].

Techniques: Stable Transfection, Expressing, Western Blot, Immunoprecipitation, Magnetic Beads

Journal: bioRxiv

Article Title: Arsenic induces two different interaction modes of SUMO with promyelocytic leukemia (PML) proteins

doi: 10.1101/2023.04.24.538205

Figure Lengend Snippet: The solubility of PML in the RIPA buffer is lost gradually and PML is SUMOylated upon exposure to As 3+ in HEK293 cells. This process appears to be completed in 2h. SUMOylated proteins including PML were pulled down by anti-RFP antibody-conjugated magnetic beads. In addition, PML and PML SUMOylated with endogenous SUMO were pulled down by via the SUMO-SIM (SUMO interacting motif) interaction in As 3+ -exposed cells. PML-VI, which does not have a SIM, is SUMOylated upon As 3+ -exposure like other PML isoforms. However, PML-VI cannot associate with SUMO2 molecules non-covalently.

Article Snippet: HEK293, which were stably transfected with the human PML-VI gene (RC220236, OriGene, Rockville, MD), were selected using neomycin and were designated as HEKPML cells [ ].

Techniques: Solubility, Magnetic Beads

Journal: bioRxiv

Article Title: Arsenic induces two different interaction modes of SUMO with promyelocytic leukemia (PML) proteins

doi: 10.1101/2023.04.24.538205

Figure Lengend Snippet: (A) Sub-confluent cells were exposed to 3 μM As 3+ (As) or left untreated (C) for 2 h before lysis with cold RIPA buffer. The lysate was separated into the RIPA-soluble (Sol) and -insoluble (Ins) fractions for the detection of DAXX, SAE1, Ubc9, and UBA2 by immunoblotting. (B) The soluble fractions of cell lyzates were used for the quantitation of DAXX in untreated HEK293, PML -/- , and HEKPML cells in triplicate wells. The electrophoresis was performed using NuPAGE 3-8% Tris-Acetate gels (Invitrogen-ThermoFisher), and two different antibodies were used for the densitometric quantitation of the clearly separated DAXX band. An open triangle indicates protein bands used for the densitometric quantification. *, Significantly different from the other two groups (N=3).

Article Snippet: HEK293, which were stably transfected with the human PML-VI gene (RC220236, OriGene, Rockville, MD), were selected using neomycin and were designated as HEKPML cells [ ].

Techniques: Lysis, Western Blot, Quantitation Assay, Electrophoresis

Journal: PLoS ONE

Article Title: Enhanced Phospholipase A2 Group 3 Expression by Oxidative Stress Decreases the Insulin-Degrading Enzyme

doi: 10.1371/journal.pone.0143518

Figure Lengend Snippet: A , B , Quantitative RT-PCR result of IDE in TR-AST cells and rat primary astrocytes. TR-AST cells and primary astrocytes were treated with hydrogen peroxide in indicated conditions. Fold changes to the non-treated cells as controls are indicated. C , Quantitative PCR results of IDE in Pla2g3 transfected HEK293 cells. Human Pla2g3 was transiently expressed and cells were harvested after 48 hours of transfection. Fold changes to the mock control are indicated. *p<0.05, ***p<0.001.

Article Snippet: HEK293 cells were used for transient transfection of human Pla2g3 expression vector (Origene).

Techniques: Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Transfection

Journal:

Article Title: Induction of Dedifferentiation, Genomewide Transcriptional Programming, and Epigenetic Reprogramming by Extracts of Carcinoma and Embryonic Stem Cells D⃞

doi: 10.1091/mbc.E05-06-0572

Figure Lengend Snippet: Morphology of 293T cells treated with NCCIT extract. (A) Untreated 293T and NCCIT cells. (B) 293T cells at indicated time points after exposure to NCCIT (a–e) or 293T (f–j) extract. (C) 293T cells 10 d after exposure to Jurkat extract and cultured under T-cell growth conditions. Dark spots are Dynal Biotech (Montebello, Norway) magnetic beads bearing antibodies against CD3 and CD28 surface antigens and used to promote T-cell expansion. Bars, 30 μm.

Article Snippet: Cells NCCIT, Jurkat (clone E6-1), and 293T cells (American Type Culture Collection, Vanassas, MD) were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) with 10% fetal calf serum (FCS), 2 mM l -glutamine, 1 mM sodium pyruvate, and nonessential amino acids (complete RPMI 1640 medium).

Techniques: Cell Culture, Magnetic Beads

Journal:

Article Title: Induction of Dedifferentiation, Genomewide Transcriptional Programming, and Epigenetic Reprogramming by Extracts of Carcinoma and Embryonic Stem Cells D⃞

doi: 10.1091/mbc.E05-06-0572

Figure Lengend Snippet: Immunofluorescence analysis of Oct4, lamin A/C and B-type lamin expression in 293T cells exposed to NCCIT extract. Untreated NCCIT and 293T cells (A) and 293T cells (B) treated with NCCIT or 293T extract were immunolabeled with antibodies against Oct4, lamin A/C, and B-type lamins (B, 1 wk after extract treatment). Bars, 20 μm. (C) Proportions (mean ± SD) of untreated NCCIT and 293T cells and of extract-treated cells expressing Oct4, lamin A/C, and B-type lamins. Three sets of 200 cells were examined for each marker. *p < 0.05 compared with 293T cells (t test); **p < 0.001 compared with 293T cells and 293T cells treated with 293T extract (t test).

Article Snippet: Cells NCCIT, Jurkat (clone E6-1), and 293T cells (American Type Culture Collection, Vanassas, MD) were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) with 10% fetal calf serum (FCS), 2 mM l -glutamine, 1 mM sodium pyruvate, and nonessential amino acids (complete RPMI 1640 medium).

Techniques: Immunofluorescence, Expressing, Immunolabeling, Marker

Journal:

Article Title: Induction of Dedifferentiation, Genomewide Transcriptional Programming, and Epigenetic Reprogramming by Extracts of Carcinoma and Embryonic Stem Cells D⃞

doi: 10.1091/mbc.E05-06-0572

Figure Lengend Snippet: Quantitative RT-PCR analysis of expression of indicated stem cell genes in 293T cells treated with NCCIT extract

Article Snippet: Cells NCCIT, Jurkat (clone E6-1), and 293T cells (American Type Culture Collection, Vanassas, MD) were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) with 10% fetal calf serum (FCS), 2 mM l -glutamine, 1 mM sodium pyruvate, and nonessential amino acids (complete RPMI 1640 medium).

Techniques: Quantitative RT-PCR, Expressing

Journal:

Article Title: Induction of Dedifferentiation, Genomewide Transcriptional Programming, and Epigenetic Reprogramming by Extracts of Carcinoma and Embryonic Stem Cells D⃞

doi: 10.1091/mbc.E05-06-0572

Figure Lengend Snippet: Oct4 expression in EGFP-labeled 293T cells. (A) 293T cells stably expressing EGFP and a geneticin resistance (GenR) gene were treated with 293T or NCCIT extract and cultured for 2 wk with 700 ng/ml geneticin before immunolabeling with anti-Oct4 antibodies. NCCIT extract was also treated with 500 μg/ml DNAse I before incubating cells (bottom row). Bar, 20 μm. (B) Quantitative RT-PCR analysis of expression of indicated genes in 293T-EGFP-GenR cells 2 wk after incubation in intact or DNAse I-treated NCCIT extract (relative to 293T extract-treated controls). (C) PCR analysis of the presence of SV40 large T antigen in 293T, NCCIT, and extract-treated cells. Ladder is a 123-base pair DNA ladder.

Article Snippet: Cells NCCIT, Jurkat (clone E6-1), and 293T cells (American Type Culture Collection, Vanassas, MD) were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) with 10% fetal calf serum (FCS), 2 mM l -glutamine, 1 mM sodium pyruvate, and nonessential amino acids (complete RPMI 1640 medium).

Techniques: Expressing, Labeling, Stable Transfection, Cell Culture, Immunolabeling, Quantitative RT-PCR, Incubation

Journal:

Article Title: Induction of Dedifferentiation, Genomewide Transcriptional Programming, and Epigenetic Reprogramming by Extracts of Carcinoma and Embryonic Stem Cells D⃞

doi: 10.1091/mbc.E05-06-0572

Figure Lengend Snippet: Bisulfite sequencing analysis of DNA methylation changes in extract-treated cells. 293T cells, NCCIT cells, and cells treated with 293T or NCCIT extract were examined for cytosine methylation in underlined CpG dinucleotides within shown genomic regions of the human OCT4 (A), LMNA (B), and LMNB1 (C) genes. Diagrams show localization (nucleotide numbers) of regions examined relative to the ATG translation start (+1).

Article Snippet: Cells NCCIT, Jurkat (clone E6-1), and 293T cells (American Type Culture Collection, Vanassas, MD) were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) with 10% fetal calf serum (FCS), 2 mM l -glutamine, 1 mM sodium pyruvate, and nonessential amino acids (complete RPMI 1640 medium).

Techniques: Methylation Sequencing, DNA Methylation Assay, Methylation

Journal:

Article Title: Induction of Dedifferentiation, Genomewide Transcriptional Programming, and Epigenetic Reprogramming by Extracts of Carcinoma and Embryonic Stem Cells D⃞

doi: 10.1091/mbc.E05-06-0572

Figure Lengend Snippet: Microarray analysis of gene expression in extract-treated 293T cells. (A) Venn diagram identifying “NCCIT-specific” genes (yellow area). Numbers of genes up- or down-regulated more than threefold (relative to input 293T cells) in cells incubated in extract of NCCIT (B), 293T (C), and Jurkat (D) cells (mean ± SD of two [B and C] and four [D] experiments). Yellow bars indicate genes up- or down-regulated in extract-treated cells and shared with NCCIT cells. In B, the likelihood that NCCIT genes are up- or down-regulated by chance rather than by extract treatment is extremely low (p < 10-5 and p < 10-4, respectively; t tests). By contrast, in C and D these probabilities are relatively high (p > 0.07 and p > 0.08, respectively; t tests). (E) Percentage of NCCIT genes up- or down-regulated in extract-treated cells (percentage of total up- or down-regulated genes). (F) Number of NCCIT genes specifically up- or down-regulated by treatment with NCCIT extract, over time. (G) Consistency of gene up- or down-regulation over time after treatment with NCCIT extract. Numbers of up- and down-regulated NCCIT genes in cells exposed to NCCIT extract are shown in green and red. Gray bars represent genes consistently up- or down-regulated at weeks 1 and 2 (gray bars at week 2), weeks 1, 2, and 4 (gray bars at week 4), etc., and shared between the two experiments. (H) Functional class distribution of genes consistently up- or down-regulated over 8 wk in two experiments (gray bars in G). These genes are listed in Table S3.

Article Snippet: Cells NCCIT, Jurkat (clone E6-1), and 293T cells (American Type Culture Collection, Vanassas, MD) were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) with 10% fetal calf serum (FCS), 2 mM l -glutamine, 1 mM sodium pyruvate, and nonessential amino acids (complete RPMI 1640 medium).

Techniques: Microarray, Gene Expression, Incubation, Functional Assay

Journal:

Article Title: Induction of Dedifferentiation, Genomewide Transcriptional Programming, and Epigenetic Reprogramming by Extracts of Carcinoma and Embryonic Stem Cells D⃞

doi: 10.1091/mbc.E05-06-0572

Figure Lengend Snippet: Quantitative RT-PCR analysis of expression of indicated multilineage priming genes in 293T cells treated with NCCIT extract relative to transcript levels in 293T cells exposed to 293T extract. Expression levels were adjusted to those of GAPDH in triplicate samples. Single data points show mean expression level in NCCIT cells.

Article Snippet: Cells NCCIT, Jurkat (clone E6-1), and 293T cells (American Type Culture Collection, Vanassas, MD) were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) with 10% fetal calf serum (FCS), 2 mM l -glutamine, 1 mM sodium pyruvate, and nonessential amino acids (complete RPMI 1640 medium).

Techniques: Quantitative RT-PCR, Expressing

Journal:

Article Title: Induction of Dedifferentiation, Genomewide Transcriptional Programming, and Epigenetic Reprogramming by Extracts of Carcinoma and Embryonic Stem Cells D⃞

doi: 10.1091/mbc.E05-06-0572

Figure Lengend Snippet: Neuronal differentiation of 293T cells treated with 293T or NCCIT extract. (A) Top, induction of differentiation. Suspended aggregates after 2 wk of culture with 10 μM all-trans-retinoic acid. Bottom, differentiation. Cells were plated onto poly-l-lysine-coated coverslips after 2 d of culture in the absence of retinoic acid but with mitotic inhibitors. Note neurite extensions in NCCIT extract-treated cells. (B) NCCIT extract-treated cells either treated with retinoic acid (+RA) or not (-RA) for 3 wk were immunolabeled using antibodies against Oct4 and nestin. Graph shows proportions (mean ± SD) of cells immunolabeled with anti-Oct4 and anti-nestin antibodies (n = 200 cells in each of a triplicate analysis for each treatment). (C) Quantitative RT-PCR analysis of expression of OCT4, NES (nestin), LMNA and LMNB1 in 293T cells treated with 293T or NCCIT extract, in absence (-RA) or presence (+RA) or retinoic acid for 3 wk. (D) NeuN and NF200 immunofluorescence analysis of indicated cell types induced to differentiate as described in A. Insets, DNA labeled with Hoechst 33342. Bars, 400 μm (A, top); 40 μm (A, bottom); 40 μm (B and D).

Article Snippet: Cells NCCIT, Jurkat (clone E6-1), and 293T cells (American Type Culture Collection, Vanassas, MD) were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) with 10% fetal calf serum (FCS), 2 mM l -glutamine, 1 mM sodium pyruvate, and nonessential amino acids (complete RPMI 1640 medium).

Techniques: Immunolabeling, Quantitative RT-PCR, Expressing, Immunofluorescence, Labeling

Journal:

Article Title: Induction of Dedifferentiation, Genomewide Transcriptional Programming, and Epigenetic Reprogramming by Extracts of Carcinoma and Embryonic Stem Cells D⃞

doi: 10.1091/mbc.E05-06-0572

Figure Lengend Snippet: Induction of adipogenic, osteogenic, and endothelial differentiation of 293T cells treated with 293T or NCCIT extract. (A and B) Cells were exposed to 10 μM retinoic acid for 21 d, washed, and cultured in the absence of retinoic acid in adipocyte (A) or osteoblast (B) differentiation medium for 21 d. (A) Cells were stained with Oil-Red-O to reveal lipid droplets. Images on the right are enlargements of two areas framed in white in the adjacent panel (top right-hand quadrant). Arrows point to strongly stained lipid droplets. (B) Cells were stained with Alzarin red to visualize mineralized nodules (arrows). Graph shows mean ± SD number of distinct strongly mineralized nodules (arrows) per unit area (area shown in B). Twenty-four to 27 areas were analyzed within wells of six-well culture plates. *p < 10-6 relative to other treatments (ANOVA). (C) 293T and NCCIT extract-treated cells were passaged onto methylcellulose for 7 d to elicit endothelial differentiation. Note the formation of a track phenotype characteristic of cultured endothelial cells (right). (D) Direct immunofluorescence labeling of CD31 and CD144 surface antigens in extract-treated cells induced to differentiate as described in C. After differentiation, cells were loosened from the methylcellulose semisolid substrate by dilution with PBS and thus lost their elongated phenotype. Bars, 40 μm (A), 200 μm (B and C), and 40 μm (D).

Article Snippet: Cells NCCIT, Jurkat (clone E6-1), and 293T cells (American Type Culture Collection, Vanassas, MD) were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) with 10% fetal calf serum (FCS), 2 mM l -glutamine, 1 mM sodium pyruvate, and nonessential amino acids (complete RPMI 1640 medium).

Techniques: Cell Culture, Staining, Immunofluorescence, Labeling

Journal: Nature Communications

Article Title: SLC13A3 is a major effector downstream of activated β-catenin in liver cancer pathogenesis

doi: 10.1038/s41467-024-51860-2

Figure Lengend Snippet: a , b mRNA expression of TBX3 , GLUL , and SLC13A3 , as well as protein levels of β-catenin and SLC13A3 in CTNNB1 -overexpressing or CTNNB1 -knockdown HCC cells. Huh7 and HLF cells were transfected with pT3-EF1αH plasmid (empty vector, EV , gray) or pT3-EF1αH plasmid containing ΔN90-β-catenin mutant fragment ( CTNNB1 , red). HepG2 and SNU398 cells were infected with shRNA lentivirus using pLKO.1 plasmid containing either scramble shRNA (negative control shRNA, sh NC , gray) or sh CTNNB1 (blue) sequences. Cells were collected at 24 h for qRT-PCR and 48 h for western blot. The experiments were performed three times on different days. Each western blot represented one biological replicate (two technical repeats per group). Data are presented as the mean ± SEM ( n = 3 independent experiments). Statistical analysis was performed using two-tailed Student’s t test. c Luciferase reporter assay for the identification of β-catenin binding sites in the SLC13A3 gene promoter region (~1.0 kb from transcription start site, TSS). A series of fragments in the SLC13A3 promoter region were schematized. HEK293T cells were transfected with the respective promoter plasmid, pCMV-renilla, and EV - or CTNNB1 -overexpressing plasmid for 24 h. Data are presented as the mean ± SEM ( n = 3 independent experiments). Statistical analysis was performed using one-way ANOVA test. d Chromatin immunoprecipitation (ChIP)-PCR detection of the SLC13A3 promoter. DNA was isolated by anti-β-catenin antibody (orange), anti-TCF4 antibody (blue), or negative control IgG (gray). Input DNA which equaled 10% total DNA samples prior to immunoprecipitation was used as positive control. DNAs were respectively amplified using SLC13A3 promoter primers#1 (for binding site 1), #2 (for binding site 2), and #3 (spans −2100 to −2081 bp upstream of the TSS), as well as negative GAPDH primers and positive MYC primers. Data are presented as the mean ± SEM ( n = 3 independent experiments). Statistical analysis was performed using two-tailed Student’s t test. e In vitro EMSA analysis of TCF4 protein binding with two putative β-catenin binding sites in HepG2 nuclear extracts. The protein and DNA interactions were abolished by adding unlabeled wild-type probes, but could not be abrogated by mutant probes. Left panel: Adding anti-TCF4 antibody resulted in a supershift band to TCF4 protein. Right panel: No supershift band after adding anti-TCF4 antibody. SLC13A3 probe #1 was for the bind site 1, and SLC13A3 probe #2 was for the bind site 2. Each experiment was independently repeated three times. f Relative intracellular levels of malate, succinate, and fumarate. The data were obtained from the untargeted metabolomics in CTNNB1 -overexpressing Huh7 cells (red) and CTNNB1 -knockdown HepG2 cells (blue). Data are presented as the mean ± SEM ( n = 6 independent experiments). Statistical analysis was performed using two-tailed Student’s t test. Source data are provided as a Source Data file.

Article Snippet: Human liver hepatocellular carcinoma cell line Huh6 (TCHu181), SK-HEP-1 (TCHu109), and human embryonic kidney cell line HEK293 (ATCC, CRL-1573) were obtained from Cell Resource Center of Shanghai Institutes for Biological Sciences (Shanghai, China).

Techniques: Expressing, Knockdown, Transfection, Plasmid Preparation, Mutagenesis, Infection, shRNA, Negative Control, Quantitative RT-PCR, Western Blot, Two Tailed Test, Luciferase, Reporter Assay, Binding Assay, Chromatin Immunoprecipitation, Isolation, Immunoprecipitation, Positive Control, Amplification, In Vitro, Protein Binding

Journal: Nature Communications

Article Title: SLC13A3 is a major effector downstream of activated β-catenin in liver cancer pathogenesis

doi: 10.1038/s41467-024-51860-2

Figure Lengend Snippet: a Cellular accumulation of 13 C 2 , 15 N-glutathione (GSH) in HEK293 cells. HEK293- SLC13A3 and HEK- EV cells were incubated with isotope-labeled GSH at concentrations from 10 nM to 10 mM for 20 min. Saturable GSH uptake was calculated using the difference of GSH accumulation in HEK293- SLC13A3 and HEK- EV cells. The data were fit to a Michaelis–Menten equation. Data are presented as the mean ± SEM for a representative experiment (n = 3 independent experiments). b Cellular uptake of 13 C 2 , 15 N-glutathione (GSH) (5 and 10 μM) was significantly higher in HEK- SLC13A3 cells (red) compared to HEK293- EV cells (gray). Cells were incubated in the uptake buffer containing GSH for 20 min. Data are presented as the mean ± SEM ( n = 3 independent experiments). Statistical analysis was performed using two-tailed Student’s t test. c Representative western blots of SLC13A3, SLC7A5, and SLC3A2 in HepG2 and SNU398 cell. Stably control (sh NC ) and SLC13A3 -knockdown (sh#1) cells were established by infection with shRNA lentivirus. Blots were representative of three independent experiments (two replicates per group for each experiment). d Cellular uptake of 2 mM leucine in HepG2 and SNU398 cells. Stability control (sh NC , gray) and SLC13A3 -knockdown cells (sh#1, blue) were incubated with 2 mM leucine for 20 min. Data are presented as the mean ± SEM ( n = 3 independent experiments). Statistical analysis was performed using two-tailed Student’s t test. e qRT-PCR and western blot analyses of MYC expression in HepG2 and SNU398 cells. Stably control (sh NC , gray) and SLC13A3 -knockdown (sh#1, blue and sh#2, dark blue) cells were established using respective shRNA lentivirus. Data are presented as the mean ± SEM ( n = 3 independent experiments). Statistical analysis was performed using one-way ANOVA test. Blots were representative of three independent experiments. f Methyl-DNA immunoprecipitation (MeDIP)-PCR detection of the MYC promoter methylation. DNA was immunoprecipitated with 5-methylcytosine antibody (blue) or IgG (gray), and purified according to the manufacturer’s protocol. Input DNA prior to immunoprecipitation was used as the positive control. Precipitated and input DNAs were amplified using primers for the CpG islands in the MYC promoter region, and PCR products were visualized by gel electrophoresis. Data are presented as the mean ± SEM ( n = 3 independent experiments). Statistical analysis was performed using two-tailed Student’s t test. g Mouse study design. Mice were given a leucine-deficient diet (Leu-) or leucine-supplemented drinking water (1.5% leucine in drinking water, Leu+). Liver tumor was induced by hydrodynamic tail vein injection (HTVi) of c-Met/β-catenin plasmids or AKT/β-catenin plasmids. h Mice were euthanized when they developed a high burden of liver tumors. The log-rank test was used to compare overall survival between groups (Gray, control; Blue, Leu+; Orange, Leu-). ns, non-statistically significant. i , j Representative gross liver images, as well as H&E (hematoxylin-eosin) and Ki67 IHC staining images. Images were representative shown out of 6 independent mice per group. Scale bar, 200 μm. Source data are provided as a Source Data file.

Article Snippet: Human liver hepatocellular carcinoma cell line Huh6 (TCHu181), SK-HEP-1 (TCHu109), and human embryonic kidney cell line HEK293 (ATCC, CRL-1573) were obtained from Cell Resource Center of Shanghai Institutes for Biological Sciences (Shanghai, China).

Techniques: Incubation, Labeling, Two Tailed Test, Western Blot, Stable Transfection, Control, Knockdown, Infection, shRNA, Quantitative RT-PCR, Expressing, Immunoprecipitation, Methylated DNA Immunoprecipitation, Methylation, Purification, Positive Control, Amplification, Nucleic Acid Electrophoresis, Injection, Immunohistochemistry

Journal: Nucleic Acids Research

Article Title: Binding of the transcription activator-like effector augments transcriptional regulation by another transcription factor

doi: 10.1093/nar/gkac454

Figure Lengend Snippet: Analysis of TALE[A] effect on reporter transcription and activity. ( A ) Nucleotide sequence of the reporter plasmid, upstream of reporter gene. All the parts changed throughout this study are annotated, the rest of the sequence remains constant. For full annotation see supplement. ( B ) Schematic representation of the luciferase reporter used in experiments C-I with (bottom) and without (top) depiction of TALE[A] and TetR:VP16 bound to their target sites. ( C ) Measurement of luciferase activity corresponding to increasing amounts of TALE[A] encoding plasmid transfected. The experiment was performed on HEK 293T cell line. ( D ) Measurement of luciferase activity on HEK 293T cell line. TALE[F] is a negative control for TALE binding to target DNA as the reporter has no binding site for TALE[F]. Where indicated, 25 ng of TALE-encoding plasmids were co-transfected. ( E ) Quantitative RT-PCR of luc2CP mRNA. Values are normalized to TetR:VP16 activation. The bars represent the mean ± s.d.; n = 3 separate experiments. Transfection mixtures of plasmids were performed as indicated in qPCR methods. Experiment was performed on HEK 293T cell line. ( F ) Measurement of luciferase activity on Neuro2A cell line. Where indicated, 25 ng of TALE-encoding plasmids were co-transfected. ( G ) Measurement of luciferase activity on CHO cell line. Where indicated, 25 ng of TALE-encoding plasmids were co-transfected. ( H ) Measurement of luciferase activity on HeLa cell line. Where indicated, 25 ng of TALE-encoding plasmids were co-transfected. ( I ) Measurement of luciferase activity on Jurkat cell line. Where indicated, 2.5 μg of TALE-encoding plasmids were co-electroporated. The bars represent the mean ± s.d.; n = 4 technical replicates. ( J ) Schematic representation of the BFP reporter used in experiment K with (bottom) and without (top) depiction of TALE[A] and TetR:VP16 bound to their targets. ( K ) Flow cytometry histogram. Percentage of BFP positive singlets is indicated next to legend. HEK 293T cells were gated to singlets and plotted by their BFP fluorescence. Transfection mixtures were performed as stated in cytometry methods. All relative luciferase units were normalized (nRLU) to luciferase activity elicited by TetR:VP16. The bars represent the mean ± s.d.; n = 4 biologically independent cell cultures, unless stated otherwise. Statistical significance between samples with an activator and with and without TALE[A] was determined by unpaired two-tailed unequal variance t -test and the P -value is specified on graph. Transfection mixtures of plasmids were performed as indicated below graph, unless stated otherwise.

Article Snippet: For flow cytometry HEK 293T cells were grown to 40–60% confluence and transfected with a mixture of jetPEI (Polyplus transfection, 3 ul/500 ng DNA, stock concentration 0.324 mg/ml, pH 7.5) and DNA.

Techniques: Activity Assay, Sequencing, Plasmid Preparation, Luciferase, Transfection, Negative Control, Binding Assay, Quantitative RT-PCR, Activation Assay, Flow Cytometry, Fluorescence, Cytometry, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Binding of the transcription activator-like effector augments transcriptional regulation by another transcription factor

doi: 10.1093/nar/gkac454

Figure Lengend Snippet: Effect of TALE and several other DNA-binding proteins on transcriptional activation. ( A ) Schematic representation of the reporters used in experiment B ( B ) Measurement of luciferase activity elicited by different TALE proteins binding to their correspondent binding site. ( C ) Schematic representation of the reporters used in experiment F ( D ) Schematic representation of the reporters used in experiment G. ( E ) Schematic representation of the reporters used in experiment H. ( F ) Measurement of luciferase activity. The added amount of Gal4-encoding plasmid is 25 ng. ( G ) Measurement of luciferase activity. The added amount of Zif268-encoding plasmid is 100 ng. ( H ) Measurement of luciferase activity. The added amount of gDNA[At] and dCas9-encoding plasmid is 50 ng each. All experiments were performed on HEK 293T cell line. Relative luciferase units were normalized (nRLU) to luciferase activity elicited by TetR:VP16. The bars represent the mean ± s.d.; n = 4 biologically independent cell cultures. Statistical significance between samples with an activator with and without TALE[A] was determined by unpaired two-tailed unequal variance t-test and the p-value is specified on graph. Transfection mixtures of plasmids were performed as indicated below each graph.

Article Snippet: For flow cytometry HEK 293T cells were grown to 40–60% confluence and transfected with a mixture of jetPEI (Polyplus transfection, 3 ul/500 ng DNA, stock concentration 0.324 mg/ml, pH 7.5) and DNA.

Techniques: DNA Binding Assay, Activation Assay, Luciferase, Activity Assay, Binding Assay, Plasmid Preparation, Two Tailed Test, Transfection

Journal: Nucleic Acids Research

Article Title: Binding of the transcription activator-like effector augments transcriptional regulation by another transcription factor

doi: 10.1093/nar/gkac454

Figure Lengend Snippet: Analysis of transcriptional activation affected by different TALE protein binding orientations and positions. ( A ) Schematic representation of the designed reverse TALE[A] binding site with bound proteins colored to illustrate the chain from the N terminal (blue) to the C terminal (red). rA is the reverse complement of the TALE[A] target site. ( B ) Nucleotide sequences of the reporter plasmids used in experiments C and D, upstream of promotor. Light blue = TALE[A] target site, dark blue = TALE[A] reverse target site, green = TetR target site, gray = constant region, magenta = spacer to ensure distance of transcription factor target site to promoter is the same for all constructs. For full annotation see supplement. ( C ) Measurement of luciferase activity examining the effect of different placements and orientations of the TALE[A] target site adjacently to TetR target site on the enhancement of transcriptional activation. Statistical significance between samples with an activator with and without TALE[A] was determined by unpaired two-tailed unequal variance t-test and the p-value is specified on graph. ( D ) Measurement of luciferase activity examining the effect TALE[A] with target sites only upstream or both upstream and downstream of the TetR:VP16 target site. Statistical significance between samples with an activator with and without TALE[A] and between samples of reporters A:tet and A:tet:A with an activator and TALE[A] was determined by unpaired two-tailed unequal variance t -test and the p-value is specified on graph. All experiments were performed on HEK 293T cell line. Transfection mixtures of plasmids were performed as indicated in each legend. Relative luciferase units were normalized (nRLU) to luciferase activity elicited by TetR:VP16. The bars represent the mean ± s.d.; n = 4 biologically independent cell cultures unless stated otherwise.

Article Snippet: For flow cytometry HEK 293T cells were grown to 40–60% confluence and transfected with a mixture of jetPEI (Polyplus transfection, 3 ul/500 ng DNA, stock concentration 0.324 mg/ml, pH 7.5) and DNA.

Techniques: Activation Assay, Protein Binding, Binding Assay, Construct, Luciferase, Activity Assay, Two Tailed Test, Transfection

Journal: Nucleic Acids Research

Article Title: Binding of the transcription activator-like effector augments transcriptional regulation by another transcription factor

doi: 10.1093/nar/gkac454

Figure Lengend Snippet: The effect of distance between target sites on TALE[A] elicited transcriptional activation ( A ) A schematic representation of the reporters used in this experiment with (bottom) and without (top) proteins bound. The length of DNA altered in this experiment is indicated in magenta. ( B ) Measurement of luciferase activity. Transfection mixtures of plasmids were performed as indicated in the legend. The experiment was performed on HEK 293T cell line. Relative luciferase units were normalized (nRLU) to luciferase activity elicited by TetR:VP16. The bars represent the mean ± s.d.; n = 4 biologically independent cell cultures. Statistical significance between samples with an activator and with and without TALE[A] was determined by unpaired two-tailed unequal variance t -test and the corresponding P -values are listed in Table S8: * P < 0.05 ** P < 0.01 *** P < 0.001, NS: P > 0.1. ( C ) The 3D model of TALE[A] (cyan) and TetR (green) bound to their respective target sites.

Article Snippet: For flow cytometry HEK 293T cells were grown to 40–60% confluence and transfected with a mixture of jetPEI (Polyplus transfection, 3 ul/500 ng DNA, stock concentration 0.324 mg/ml, pH 7.5) and DNA.

Techniques: Activation Assay, Luciferase, Activity Assay, Transfection, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Binding of the transcription activator-like effector augments transcriptional regulation by another transcription factor

doi: 10.1093/nar/gkac454

Figure Lengend Snippet: Effect of TALE on transcriptional activation in combination with different types of transcription factors. ( A ) Schematic representation of the reporters used in experiment B, with (bottom) and without (top) proteins bound. The length of DNA altered in this experiment is indicated in magenta. ( B ) Measurement of luciferase activity elicited by the effect of TALE[A] on Gal4:VP16. ( C ) 3D model of the TALE[A] dsDNA Gal4 complex shown in cyan-grey-pink, respectively. The red surface represents a steric obstruction. The TALE[A] surface is partially transparent, so the red surface is better visible. Sequence labels and spacers are indicated below each model. ( D ) Schematic representation of the reporters used in experiment E, with (bottom) and without (top) bound proteins. The length of DNA altered in this experiment is indicated in magenta. ( E ) Measurement of luciferase activity elicited by the effect of TALE[A] on Zif268:VP16. ( F ) Model of TALE[A] dsDNA Zif268 complex shown in cyan-grey-yellow, respectively. The red surface represents the steric obstruction. The TALE[A] surface is partially transparent, so the red surface is better visible. ( G ) Model of the TALE[A] dsDNA Zif268:VP16 complex shown in cyan-grey-yellow, respectively. The red surface represents the steric obstruction. The TALE[A] surface is partially transparent, so the red surface is better visible. Sequence labels and spacers are indicated below each model. All experiments were performed on HEK 293T cell line. Transfection mixtures of plasmids were performed as indicated in legends. Relative luciferase units were normalized (nRLU) to luciferase activity elicited by activator. The bars represent the mean ± s.d.; n = 4 biologically independent cell cultures. Statistical significance between samples with and without TALE[A] was determined by unpaired two-tailed unequal variance t-test and the corresponding p-values are listed in Table S9 and S10 for B and E, respectively. * P < 0.05, ** P < 0.01, *** P < 0.001, NS: P > 0.1.

Article Snippet: For flow cytometry HEK 293T cells were grown to 40–60% confluence and transfected with a mixture of jetPEI (Polyplus transfection, 3 ul/500 ng DNA, stock concentration 0.324 mg/ml, pH 7.5) and DNA.

Techniques: Activation Assay, Luciferase, Activity Assay, Sequencing, Transfection, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Binding of the transcription activator-like effector augments transcriptional regulation by another transcription factor

doi: 10.1093/nar/gkac454

Figure Lengend Snippet: Effect of TALE on augmentation of transcriptional repression. ( A ) Nucleotide sequence of the reporter plasmid, upstream of reporter gene. All the parts changed throughout this experiment are annotated, the rest of the sequence remains constant. For full annotation see supplement. ( B ) Schematic representation of the reporters used in experiment E. with (bottom) and without (top) proteins bound. ( C ) Schematic representation of the reporters used in experiment F. with (bottom) and without (top) proteins bound. ( D ) Schematic representation of the reporters used in experiment G. with (bottom) and without (top) proteins bound. ( E ) Measurement of luciferase activity elicited by the effect of TALE[A] on TetR:KRAB. ( F ) Measurement of luciferase activity elicited by the effect of TALE[A] on Gal4:KRAB. ( G ) Measurement of luciferase activity elicited by the effect of TALE[A] on Zif268:VP16. All experiments were performed on HEK 293T cell line. Transfection mixtures of plasmids were performed as indicated in legends, where indicated, 25 ng of TALE[A] encoding plasmid was co transfected. Relative luciferase units were normalized (nRLU) to luciferase activity elicited by reporter alone. The bars represent the mean ± s.d.; n = 4 biologically independent cell cultures. Statistical significance between samples with and without TALE[A] was determined by unpaired two-tailed unequal variance t-test and the P -value is specified on graph.

Article Snippet: For flow cytometry HEK 293T cells were grown to 40–60% confluence and transfected with a mixture of jetPEI (Polyplus transfection, 3 ul/500 ng DNA, stock concentration 0.324 mg/ml, pH 7.5) and DNA.

Techniques: Sequencing, Plasmid Preparation, Luciferase, Activity Assay, Transfection, Two Tailed Test

Journal: iScience

Article Title: ALKBH5-mediated m 6 A demethylation of HS3ST3B1-IT1 prevents osteoarthritis progression

doi: 10.1016/j.isci.2023.107838

Figure Lengend Snippet: ALKBH5 increases HS3ST3B1-IT1 expression through an m 6 A-YTHDF2-dependent mechanism (A) Dot blot analyses with an anti-m 6 A antibody using normal and osteoarthritic cartilages. Methylene blue (MB) staining served as a loading control (n = 5). (B) The m 6 A modification sites in HS3ST3B1-IT1 were predicted with the online tool SRAMP ( http://www.cuilab.cn/sramp ). (C) The m 6 A level of HS3ST3B1-IT1 in chondrocytes was determined by MeRIP assay. The fold enrichment of HS3ST3B11-IT1 in m 6 A pellet was relative to its matching IgG control. Values were shown as mean ± SD, n = 3 biologically independent samples. (D) qRT-PCR was performed to detect the expression levels of ALKBH5 and HS3ST3B1-IT1 in chondrocytes after transfection with ALKBH5 expression plasmid. Values were shown as mean ± SD (n = 3). (E) The expression levels of ALKBH5 in normal and osteoarthritic cartilage were evaluated by qRT-PCR (n = 50). β-actin served as an internal control. n = 50 normal cartilage tissues from 50 individuals with no prior medical history of OA and undergoing hip arthroplasty surgery, and n = 50 OA cartilages from 50 patients with OA undergoing total knee replacement surgery. Each dot represented one donor as calculated by log 2 transform. The horizontal lines indicated the mean ± SD from different donors per group. (F) Associations between ALKBH5 and HS3ST3B1-IT1 in osteoarthritic cartilage (n = 50) were assessed by Pearson correlation analysis. (G) The m 6 A level of HS3ST3B1-IT1 was evaluated by MeRIP assay in chondrocytes after transfecting with ALKBH5 expression plasmid. Values were shown as mean ± SD, n = 3 biologically independent samples. (H) The chondrocytes were treated with Act D for the indicated times, and qRT-PCR was performed to evaluate the expression level of HS3ST3B1-IT1 upon ALKBH5 overexpression. (I) Left panel: the wild-type (wt) and m 6 A consensus sequence mutant (mut) HS3ST3B1-IT1 fused with firefly luciferase reporter. Right panel: luciferase reporter assay in HEK 293T cells transfected with luciferase reporter constructs (wt or mut) and ALKBH5 expression plasmid. Values were shown as mean ± SD, n = 3 biologically independent samples. (J) RIP assay was performed in chondrocytes with anti-YTHDF2 antibody, and the coprecipitated RNAs were subjected to detect HS3ST3B1-IT1 using qRT-PCR. The fold enrichment of HS3ST3B1-IT1 in YTHDF2 pellet is relative to its matching IgG control. Values were shown as mean ± SD, n = 3 biologically independent samples. (K) The chondrocytes were transfected with YTHDF2 wild type or mutant expression plasmids and then treated with Act D for the indicated times. qRT-PCR was performed to evaluate the expression level of HS3ST3B1-IT1. Values were shown as mean ± SD (n = 3). Statistical analysis was performed using an unpaired Student’s two-tailed t test (C–E, G, I–J). OA, osteoarthritis; MB, methylene blue; wt, wild-type; mut, mutant.

Article Snippet: HEK 293T cells , ATCC , Cat# CRL-11268.

Techniques: Expressing, Dot Blot, Staining, Control, Modification, Quantitative RT-PCR, Transfection, Plasmid Preparation, Over Expression, Sequencing, Mutagenesis, Luciferase, Reporter Assay, Construct, Two Tailed Test

Journal: iScience

Article Title: ALKBH5-mediated m 6 A demethylation of HS3ST3B1-IT1 prevents osteoarthritis progression

doi: 10.1016/j.isci.2023.107838

Figure Lengend Snippet:

Article Snippet: HEK 293T cells , ATCC , Cat# CRL-11268.

Techniques: Recombinant, Virus, Control, Radio Immunoprecipitation, Membrane, SYBR Green Assay, Cell Counting, Luciferase, Reporter Gene Assay, Mutagenesis, Immunohistochemical staining, Plasmid Preparation, DNA Extraction, TUNEL Assay, Apoptosis Assay, Software