Journal: Cell reports

Article Title: PRMT6 Regulates RAS/RAF Binding and MEK/ERK-Mediated Cancer Stemness Activities in Hepatocellular Carcinoma through CRAF Methylation.

doi: 10.1016/j.celrep.2018.09.053

Figure Lengend Snippet: Figure 1. PRMT6 Is Weakly Expressed and Negatively Correlated with Aggressive Cancer Features in HCC Patients (A and B) Scattered dot (A) and waterfall (B) plot analyses of PRMT6 mRNA levels in HCC and matched non-tumor liver specimens from 77 patient samples. Red and blue bars represent samples that show a relative PRMT6 fold change of R2 overexpression and underexpression, respectively (HCC/NT). (C) Scattered dot plot analysis of PRMT6 mRNA levels in normal (n = 50) and HCC (n = 371) tissues using information gathered from the TCGA HCC dataset. (D) PRMT6 immunostaining of tissue microarray comprising 83 paired non-tumor liver and HCC tissue samples. Shown are representative images of the immunostaining. Scale bar, 100 mm. (E) Graph indicates the percentage of cases displaying no and/or low or moderate and/or high staining intensity of PRMT6. (F) Proteomic expression of PRMT6 in HCC cell lines by western blot. Data are representative of two or more independent experiments. Bars and error represent mean ± SD of replicate measurements.

Article Snippet: Sections were subsequently incubated with anti-human/mouse PRMT6 (1:1000 for human, 1:100 for mouse, Abcam, ab47244), anti-human PRMT6 (1:300, Atlas Antibodies, HPA059424), anti-mouse SOX2 (1:100, R&D Systems, MAB2018) and anti-mouse p-ERK1/2 (1:100, Abcam, ab5011).

Techniques: Over Expression, Immunostaining, Microarray, Staining, Expressing, Western Blot

Journal: Cell reports

Article Title: PRMT6 Regulates RAS/RAF Binding and MEK/ERK-Mediated Cancer Stemness Activities in Hepatocellular Carcinoma through CRAF Methylation.

doi: 10.1016/j.celrep.2018.09.053

Figure Lengend Snippet: Figure 2. PRMT6 Negatively Regulates Cancer Properties in HCC (A) Representative images and quantification of number of cells that migrated and invaded in BEL7402 and Huh7 cells with or without PRMT6 expression modulated. **p < 0.01 and ***p < 0.001. Scale bar, 100 mm. (B) Percentage of annexin V-PI-positive cells in BEL7402 and Huh7 cells with or without PRMT6 expression modulated in the presence of 5-flurouracil, cisplatin, or sorafenib. (C) Xenograft tumors and tumor volume measurements of Huh7 cells with or without PRMT6 overexpressed. (D) Bioluminescence imaging and luciferase signal quantification of nude mice injected intrahepatically with luciferase-labeled BEL7402 cells with or without PRMT6 suppressed. (E) In vivo bioluminescence imaging of nude mice injected intrahepatically with luciferase-labeled MHCC97L cells with or without PRMT6 overexpressed. Ex vivo imaging of the lungs harvested. Representative H&E images of liver and lung tissues harvested. Scale bar, 100 mm. Bar chart summary of number of metastatic foci observed in lung. EV, empty vector control; NTC, non-target control; OE, overexpression. Data are representative of two or more independent experiments. Bars and error represent mean ± SD of replicate measurements.

Article Snippet: Sections were subsequently incubated with anti-human/mouse PRMT6 (1:1000 for human, 1:100 for mouse, Abcam, ab47244), anti-human PRMT6 (1:300, Atlas Antibodies, HPA059424), anti-mouse SOX2 (1:100, R&D Systems, MAB2018) and anti-mouse p-ERK1/2 (1:100, Abcam, ab5011).

Techniques: Expressing, Imaging, Luciferase, Injection, Labeling, In Vivo, Ex Vivo, Plasmid Preparation, Control, Over Expression

Journal: Cell reports

Article Title: PRMT6 Regulates RAS/RAF Binding and MEK/ERK-Mediated Cancer Stemness Activities in Hepatocellular Carcinoma through CRAF Methylation.

doi: 10.1016/j.celrep.2018.09.053

Figure Lengend Snippet: Figure 3. PRMT6 Negatively Regulates Cancer Stemness Properties in HCC (A) Oncosphere formation and serial passages of BEL7402 and Huh7 cells with or without PRMT6 expression modulated. ***p < 0.001. Scale bar, 100 mm. (B) Flow cytometry and western blot analyses for CD133 expression in Huh7 cells with or without PRMT6 overexpressed. (C) Western blot analysis for PRMT6, SOX2, and NANOG expression in BEL7402 and Huh7 cells with or without PRMT6 expression modulated. (D) Images of xenograft tumors (red arrows) formed in NOD/SCID mice injected subcutaneously with CD133+PRMT6low, CD133+PRMT6high, CD133PRMT6low, and CD133-PRMT6high cells isolated from Huh7 cells in primary passage (picture representative of 10,000 cells injected). Kaplan-Meier curves showing percentage of tumor-free survival of the annotated groups of primary and secondary recipient mice. n = 8. Engraftment rates, average tumor latency, and tumor- initiating frequency of CD133+ and CD133 subsets with or without PRMT6 overexpressed in Huh7 cells. EV, empty vector control; NTC, non-target control; OE, overexpression; TIC, tumor-initiating cells. Data are representative of two or more independent experiments. Bars and error represent mean ± SD of replicate measurements.

Article Snippet: Sections were subsequently incubated with anti-human/mouse PRMT6 (1:1000 for human, 1:100 for mouse, Abcam, ab47244), anti-human PRMT6 (1:300, Atlas Antibodies, HPA059424), anti-mouse SOX2 (1:100, R&D Systems, MAB2018) and anti-mouse p-ERK1/2 (1:100, Abcam, ab5011).

Techniques: Expressing, Flow Cytometry, Western Blot, Injection, Isolation, Plasmid Preparation, Control, Over Expression

Journal: Cell reports

Article Title: PRMT6 Regulates RAS/RAF Binding and MEK/ERK-Mediated Cancer Stemness Activities in Hepatocellular Carcinoma through CRAF Methylation.

doi: 10.1016/j.celrep.2018.09.053

Figure Lengend Snippet: Figure 4. The Catalytically Active Domain of PRMT6 Is Functionally Important in Contributing Augmented Aggressive Cancer Stemness Properties in HCC (A) Western blot analysis for ADMA expression in BEL7402 and Huh7 cells with or without PRMT6 expression modulated. (B) Schematic illustration of PRMT6 wild-type (WT) and PRMT6 catalytic inactive (mutant) constructs used in this study. (C–E) Representative images and quantification of number of cells that (C) migrated, (D) invaded, and (E) formed oncospheres in BEL7402 cells stably over- expressing EV control, WT, and mutant PRMT6. **p < 0.01 and ***p < 0.001. Scale bar, 100 mm. (F)PercentageofannexinV-PI-positivecells inBEL7402cellsstably overexpressingEVcontrol,WT,andmutantinthe presenceof5-flurouracil,cisplatin,orsorafenib. (G) Western blot analysis for PRMT6, SOX2, and NANOG expression in BEL7402 cells stably overexpressing EV control, WT, and mutant PRMT6. (H) In vivo bioluminescence imaging and luciferase signal quantification of nude mice injected intrahepatically with luciferase-labeled BEL7402 cells stably overexpressing EV control, WT, and mutant PRMT6. EV, empty vector control; mut, mutant; NTC, non-target control; OE, overexpression; WT, wild-type. Data are representative of two or more independent experiments. Bars and error represent mean ± SD of replicate measurements.

Article Snippet: Sections were subsequently incubated with anti-human/mouse PRMT6 (1:1000 for human, 1:100 for mouse, Abcam, ab47244), anti-human PRMT6 (1:300, Atlas Antibodies, HPA059424), anti-mouse SOX2 (1:100, R&D Systems, MAB2018) and anti-mouse p-ERK1/2 (1:100, Abcam, ab5011).

Techniques: Western Blot, Expressing, Mutagenesis, Construct, Stable Transfection, Control, In Vivo, Imaging, Luciferase, Injection, Labeling, Plasmid Preparation, Over Expression

Journal: Cell reports

Article Title: PRMT6 Regulates RAS/RAF Binding and MEK/ERK-Mediated Cancer Stemness Activities in Hepatocellular Carcinoma through CRAF Methylation.

doi: 10.1016/j.celrep.2018.09.053

Figure Lengend Snippet: Figure 5. PRMT6 Interacts Directly with CRAF and Methylates It on Arginine 100 Residue (A) Gene set enrichment analysis (GSEA) identified an enrichment of RAS signaling in PRMT6 silenced cells. (B) Co-immunoprecipitation analysis for validation of CRAF as a PRMT6 interacting protein partner in 293T cells expressing SFB-tagged PRMT6, 293T cells expressing SFB-tagged CRAF, BEL7402 cells expressing SFB-tagged PRMT6, and BEL7402 expressing endogenous PRMT6. (C) Left: western blot analysis for PRMT6-mediated incorporation of asymmetric arginine dimethylation in CRAF. Middle: western blot analysis of in vitro methylation assay of CRAF. Right: GAR (glycine-arginine-rich) sequence positive control in the in vitro methylation assay. (D) Schematic diagram illustrating of full-length (FL) and deletion mutations of CRAF used in this study. CRD, cysteine-rich domain; RBD, Ras binding domain. (E) Mapping of protein domain in CRAF methylated by PRMT6 through in vivo methylation assay. Red arrows indicate ADMA bands at the predicted size. White arrow indicates loss of ADMA band at the predicted size in D2 CRAF truncation mutant. Yellow arrows indicate successful FLAG pull-down. (F) Left: levels of ADMA in WT and site-directed mutants of CRAF. Right: in vitro methylation assay of CRAF R100K mutant versus WT. (G) Left: in vitro methylation assay with immunoprecipitated PRMT6 from 293T cells stably transfected with SFB-PRMT6. Assay was performed by adding no peptide, CAVFRLLHE peptide, or CAVFKLLHE mutant peptide. Right: fragmentation spectrum of the methylated peptide identified by liquid chromatography/ tandem mass spectrometry (LC-MS/MS). m/z, mass/charge ratio. ***p < 0.001. Endo, endogenous; Exo, exogenous; KD, knockdown; mut, mutant; NTC, non-target control; WT, wild-type. Data are representative of two or more independent experiments. Bars and error represent mean ± SD of replicate measurements.

Article Snippet: Sections were subsequently incubated with anti-human/mouse PRMT6 (1:1000 for human, 1:100 for mouse, Abcam, ab47244), anti-human PRMT6 (1:300, Atlas Antibodies, HPA059424), anti-mouse SOX2 (1:100, R&D Systems, MAB2018) and anti-mouse p-ERK1/2 (1:100, Abcam, ab5011).

Techniques: Residue, Immunoprecipitation, Biomarker Discovery, Expressing, Western Blot, In Vitro, Methylation, Sequencing, Positive Control, Binding Assay, In Vivo, Mutagenesis, Stable Transfection, Transfection, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Knockdown, Control

Journal: Cell reports

Article Title: PRMT6 Regulates RAS/RAF Binding and MEK/ERK-Mediated Cancer Stemness Activities in Hepatocellular Carcinoma through CRAF Methylation.

doi: 10.1016/j.celrep.2018.09.053

Figure Lengend Snippet: Figure 6. PRMT6 Methylation of CRAF at Arginine 100 Interferes with RAS/RAF Binding Domain and Inhibits MEK/ERK-Related Kinase Activity (A) CRAF kinase assay, western blot analysis for expression of phosphorylated and total MEK1/2, phosphorylated and total ERK1/2 and ERK kinase assay in BEL7402 and Huh7 cells with or without PRMT6 expression modulated. (B) Representative images and quantification of number of cells that migrated, invaded, and formed oncospheres in BEL7402 cells expressing NTC or shPRMT6 clone 956 that were treated with control or MEK inhibitor U0126. ***p < 0.001. Scale bar, 100 mm. (C) Percentage of annexin V-PI-positive cells in BEL7402 cells expressing NTC or shPRMT6 clone 956 that were treated with control or MEK inhibitor U0126, following sorafenib treatment. (D) Western blot analysis for expression of CRAF, phosphorylated and total ERK1/2, as well as ERK kinase assay in BEL7402 cells with EV control, CRAF WT, or CRAF R100K overexpressed. (E and F) Western blot analysis for the co-immunoprecipitation of CRAF and RAS in BEL7402 cells (E) with or without PRMT6 expression modulated and (F) with CRAF WT or R100K mutant. (G) In vitro methylation assay of CRAF WT and R100K mutant followed by RAS binding assay with immunoprecipitated MBP-RAS. EV, empty vector control; NTC, non-target control; OE, overexpression; WT, wild-type. Data are representative of two or more independent experiments. Bars and error represent mean ± SD of replicate measurements.

Article Snippet: Sections were subsequently incubated with anti-human/mouse PRMT6 (1:1000 for human, 1:100 for mouse, Abcam, ab47244), anti-human PRMT6 (1:300, Atlas Antibodies, HPA059424), anti-mouse SOX2 (1:100, R&D Systems, MAB2018) and anti-mouse p-ERK1/2 (1:100, Abcam, ab5011).

Techniques: Methylation, Binding Assay, Activity Assay, Kinase Assay, Western Blot, Expressing, Control, Immunoprecipitation, Mutagenesis, In Vitro, Plasmid Preparation, Over Expression

Journal: Cell reports

Article Title: PRMT6 Regulates RAS/RAF Binding and MEK/ERK-Mediated Cancer Stemness Activities in Hepatocellular Carcinoma through CRAF Methylation.

doi: 10.1016/j.celrep.2018.09.053

Figure Lengend Snippet: Figure 7. Loss of PRMT6 Expression Aggravates Liver Tumorigenesis in a DEN+CCL4 HCC-Induced PRMT6 Knockout Mouse Model, and PRMT6-Dependent CRAF/ERK Signaling Regulates HCC Stemness as Demonstrated in Patient-Derived Organoids (A) Representative pictures of livers harvested from WT and PRMT6 knockout (PRMT6/) mice that received DEN+CCl4 carcinogen induction. (B) Maximum size of HCC tumors and liver weight over body weight ratio. *p < 0.05. (C) Representative H&E and IHC images of PRMT6 and p-ERK1/2 expression of liver tissues harvested from WT or PRMT6/ mice treated with DEN+CCL4. Scale bar, 100 mm (inset, 20 mm). (D) Western blot analysis for expression of PRMT6, CD133, NANOG, SOX2, and phosphorylated and total ERK1/2 in non-tumor liver and HCC organoids with PRMT6 stably suppressed or overexpressed, respectively. (E) Percentage of viable cells in non-tumor liver organoids with PRMT6 suppressed, compared with controls, following 5-flurouracil, cisplatin, and sorafenib treatment. *p < 0.05, **p < 0.01, and ***p < 0.001. (F) Representative images and quantification of number of cells that migrated, invaded, and formed oncospheres in HCC organoids with PRMT6 overexpressed compared with controls. Scale bar, 100 mm. *p < 0.05, **p < 0.01, and ***p < 0.001. EV, empty vector control; NT, non-tumor liver; NTC, non-target control; OE, overexpression; T, tumor/HCC. Data are representative of two or more independent experiments. Bars and error represent mean ± SD of replicate measurements.

Article Snippet: Sections were subsequently incubated with anti-human/mouse PRMT6 (1:1000 for human, 1:100 for mouse, Abcam, ab47244), anti-human PRMT6 (1:300, Atlas Antibodies, HPA059424), anti-mouse SOX2 (1:100, R&D Systems, MAB2018) and anti-mouse p-ERK1/2 (1:100, Abcam, ab5011).

Techniques: Expressing, Knock-Out, Derivative Assay, Western Blot, Stable Transfection, Plasmid Preparation, Control, Over Expression

Journal: Virology Journal

Article Title: Overexpression of PRMT6 does not suppress HIV-1 Tat transactivation in cells naturally lacking PRMT6

doi: 10.1186/1743-422X-10-207

Figure Lengend Snippet: The termini of PRMT6 are required for the interaction with HIV-1 Tat. ( A ) Upper panel , scale representation of PRMT6 protein showing amino acid positions 1 and 375, the catalytic domain (gray), and the signature methyltransferase motifs (I, I ′ , II and III; black bars). Lower panel , scale representations of Myc epitope-tagged PRMT6 (Myc-PRMT6) protein and derivative domain deletion mutants. The numbers above each schema represent the amino acid boundaries of the domain deletions. The amino-terminal Myc epitope tag is represented as a vertical bar. ( B ) Interactions between FLAG epitope-tagged Tat (Tat-FLAG) and wild type or domain deleted Myc-PRMT6 as determined by immunoprecipitation. HeLa cells were transfected to express Tat-FLAG with either wild type Myc-PRMT6 (WT) or a domain deletion mutant of Myc-PRMT6. Immunoprecipitations were performed on lysates prepared from transfected cells using anti-FLAG agarose beads (αFLAG IP). Cell lysates (left panel) and immunoprecipitates (right panel) were western blotted using anti-Myc and anti-FLAG antibodies. Loading of cell lysates was normalized for equal amounts of co expressed Renilla luciferase in each sample. The Myc-PRMT6 domain deletion mutant designations are as in A . Data are representative of three independent experiments.

Article Snippet: Endogenous PRMT6 was detected with rabbit anti-PRMT6 antibody (LifeSpan Biosciences).

Techniques: FLAG-tag, Immunoprecipitation, Transfection, Mutagenesis, Western Blot, Luciferase

Journal: Virology Journal

Article Title: Overexpression of PRMT6 does not suppress HIV-1 Tat transactivation in cells naturally lacking PRMT6

doi: 10.1186/1743-422X-10-207

Figure Lengend Snippet: The activation domain of Tat is required for the interaction with PRMT6. ( A ) Upper panel , scale representation of the two exon-encoded HIV-1 Tat protein showing amino acid positions 1 and 101, functional domains (italicized text) and structural regions (bold text) as defined by . ARM, arginine rich motif; Aux., auxiliary region. Lower panel , scale representations of FLAG epitope-tagged Tat (Tat-FLAG) protein and derivative domain deletion mutants. The numbers above each schema represent the amino acid boundaries of the domain deletions. The carboxyl-terminal FLAG epitope tag is represented as a vertical bar. ( B ) Interactions between Myc epitope-tagged PRMT6 (Myc-PRMT6) and wild type or domain deleted Tat-FLAG as determined by immunoprecipitation. HeLa cells were transfected to express Myc-PRMT6 with either wild type Tat-FLAG (WT) or a domain deletion mutant of Tat-FLAG. Immunoprecipitations were performed on lysates prepared from transfected cells using anti-FLAG agarose beads (αFLAG IP). Cell lysates (left panel) and immunoprecipitates (right panel) were western blotted using anti-Myc and anti-FLAG antibodies. Loading of cell lysates was normalized for equal amounts of co expressed Renilla luciferase in each sample. The Tat-FLAG domain deletion mutant designations are as in A . Data are representative of three independent experiments.

Article Snippet: Endogenous PRMT6 was detected with rabbit anti-PRMT6 antibody (LifeSpan Biosciences).

Techniques: Activation Assay, Functional Assay, FLAG-tag, Immunoprecipitation, Transfection, Mutagenesis, Western Blot, Luciferase

Journal: Virology Journal

Article Title: Overexpression of PRMT6 does not suppress HIV-1 Tat transactivation in cells naturally lacking PRMT6

doi: 10.1186/1743-422X-10-207

Figure Lengend Snippet: Acidic and cysteine residues within the Tat activation domain are required for interaction with PRMT6. ( A ) Schematic representation of the Tat activation domain from amino acids 1 to 37 and the basic domain from amino acids 49 to 57. The wild type (WT) amino acid sequence is shown using the single-letter amino acid code. Sequences of the acidic residues mutant (EDE), the cysteine residues mutant (CS) and the basic domain mutant (NB) are also shown, with substitution mutations indicated in reverse video. ARM, arginine rich motif. ( B ) Interactions between Myc epitope-tagged PRMT6 (Myc-PRMT6) and wild type, EDE mutant or CS mutant FLAG epitope-tagged Tat (Tat-FLAG) as determined by immunoprecipitation. HeLa cells were transfected to express Myc-PRMT6 with either wild type Tat-FLAG (WT) or one of its mutants as indicated. Immunoprecipitations were performed on lysates prepared from transfected cells using anti-FLAG agarose beads (αFLAG IP). Cell lysates (left panel) and immunoprecipitates (right panel) were western blotted using anti-Myc and anti-FLAG antibodies. ( C ) The same experiment as in B was performed with the NB mutant of Tat-FLAG. Loading of cell lysates was normalized for equal amounts of co expressed Renilla luciferase in each sample. All data are representative of three independent experiments.

Article Snippet: Endogenous PRMT6 was detected with rabbit anti-PRMT6 antibody (LifeSpan Biosciences).

Techniques: Activation Assay, Sequencing, Mutagenesis, FLAG-tag, Immunoprecipitation, Transfection, Western Blot, Luciferase

Journal: Virology Journal

Article Title: Overexpression of PRMT6 does not suppress HIV-1 Tat transactivation in cells naturally lacking PRMT6

doi: 10.1186/1743-422X-10-207

Figure Lengend Snippet: Expressed sequence tag data (shown as transcripts per million) for the PRMT6 and PRMT1 genes in selected tissue types

Article Snippet: Endogenous PRMT6 was detected with rabbit anti-PRMT6 antibody (LifeSpan Biosciences).

Techniques: Sequencing

Journal: Virology Journal

Article Title: Overexpression of PRMT6 does not suppress HIV-1 Tat transactivation in cells naturally lacking PRMT6

doi: 10.1186/1743-422X-10-207

Figure Lengend Snippet: The A549 cell line does not express detectable levels of PRMT6 protein. ( A ) Western blot of cell lysates from the A549, BJAB and HeLa cell lines detected with anti-PRMT6, anti-PRMT1 and anti-β-tubulin antibodies as indicated. ( B ) Relative expression ratios of PRMT1 and PRMT6 mRNA transcripts in A549 versus HeLa cells. Total RNA were extracted from A549 and HeLa cells before being reverse transcribed into cDNA using random primers. Quantitative PCR was then performed on the cDNA samples using primers specific for PRMT1 , PRMT6 and GAPDH transcripts. Relative expression ratios for PRMT1 and PRMT6 were calculated according to the method of . ( C ) Ectopic Myc-PRMT6 increases the steady state levels of Tat-FLAG protein in A549 cells. Western blotting was performed on A549 cells transfected to express Tat-FLAG with (+) or without (-) Myc-PRMT6. Proteins were detected with anti-FLAG and anti-PRMT6 antibodies, respectively. Loading of cell lysates was normalized for equal amounts of co expressed Renilla luciferase in each sample. ( D ) A549 cells transfected to express Tat-FLAG with or without Myc-PRMT6 were treated with cycloheximide and harvested at 0, 1, 2, 4, 6 and 8 h post-treatment. Western blotting was performed on total protein-equalized lysates. β-tubulin levels demonstrate equal sample loadings. ( E ) The Tat-FLAG band intensities in panel D were quantified, and their natural log values were plotted as a function of time. Values for Tat-FLAG co expressed with Myc-PRMT6 are indicated by the black boxes and values for Tat-FLAG expressed alone by the white boxes. The calculated Tat-FLAG protein half-lives are shown in the inset.

Article Snippet: Endogenous PRMT6 was detected with rabbit anti-PRMT6 antibody (LifeSpan Biosciences).

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction, Transfection, Luciferase

Journal: Virology Journal

Article Title: Overexpression of PRMT6 does not suppress HIV-1 Tat transactivation in cells naturally lacking PRMT6

doi: 10.1186/1743-422X-10-207

Figure Lengend Snippet: Overexpression of Myc-PRMT6 does not affect basal transcription from the HIV-1 LTR promoter. A549 cells were transfected with a long terminal repeat (LTR)- Photinus luciferase transcriptional reporter plasmid with varying amounts of Myc-PRMT6 plasmid, as indicated. All samples were cotransfected with a SV40-promotered Renilla luciferase plasmid to control for transfection efficiency variations. Transfected cells were lysed and assayed for both Photinus and Renilla luciferase activities, the ratio of which is shown for each sample. Columns represent the means and standard deviations of three independent experiments.

Article Snippet: Endogenous PRMT6 was detected with rabbit anti-PRMT6 antibody (LifeSpan Biosciences).

Techniques: Over Expression, Transfection, Luciferase, Plasmid Preparation

Journal: Virology Journal

Article Title: Overexpression of PRMT6 does not suppress HIV-1 Tat transactivation in cells naturally lacking PRMT6

doi: 10.1186/1743-422X-10-207

Figure Lengend Snippet: Overexpression of Myc-PRMT6 does not alter Tat-FLAG mediated transactivation in either A549 or HeLa cells. ( A ) A549 cells were transfected with a long terminal repeat (LTR)- Photinus luciferase transactivation reporter plasmid along with varying amounts of Tat-FLAG plasmid, as indicated, and either with (black columns) or without (white columns) Myc-PRMT6 plasmid. All samples were cotransfected with a SV40-promotered Renilla luciferase plasmid to control for transfection efficiency and assay variations. Transfected cells were lysed and assayed for both Photinus and Renilla luciferase activities, the ratio of which is shown for each sample expressed relative to the “0 ng Tat-FLAG” sample. Columns represent the means and standard deviations of four independent experiments. ( B ) The same experiment as in A was performed in HeLa cells. Columns represent the means and standard deviations of three independent experiments. ( C ) Overexpression of PRMT6 does not alter overall plasmid expression. The Renilla luciferase activities from A were normalized to total lysate protein concentrations before being expressed relative to the “0 ng Tat-FLAG without Myc-PRMT6” sample. Samples in which Myc-PRMT6 plasmid was (+) or was not (-) cotransfected are indicated. Columns represent the means and standard deviations of four independent experiments. ( D ) The same determinations as in C were performed with the samples from B . Columns represent the means and standard deviations of three independent experiments.

Article Snippet: Endogenous PRMT6 was detected with rabbit anti-PRMT6 antibody (LifeSpan Biosciences).

Techniques: Over Expression, Transfection, Luciferase, Plasmid Preparation, Expressing

Journal: Virology Journal

Article Title: Overexpression of PRMT6 does not suppress HIV-1 Tat transactivation in cells naturally lacking PRMT6

doi: 10.1186/1743-422X-10-207

Figure Lengend Snippet: HIV-1 produced in A549 cells overexpressing Myc-PRMT6 are competent for infectivity. ( A ) A549 cells were transfected with a HIV-1 proviral plasmid in which the EGFP gene had been inserted into the nef reading frame, along with a plasmid expressing the VSV-G envelope glycoprotein to enable pseudotyping. Cells were simultaneously cotransfected with either empty vector (-), the wild type Myc-PRMT6 plasmid (WT) or a methyltransferase-inactive mutant Myc-PRMT6 plasmid (Mut.). Western blotting was performed on producer cell lysates using an anti-PRMT6 antibody. Loading of cell lysates was normalized for equal amounts of co expressed Renilla luciferase in each sample. ( B ) HeLa and A549 target cells were infected with the pseudotyped viruses collected from the A549 producer cells in A after normalizing for capsid levels. EGFP expression in the target cells, which indicates successful infection, was then quantified by flow cytometry. A representative dot plot of target cells infected with virus from the empty vector-cotransfected producer cells is shown, in which GFP-positive, phycoerythrin (PE)-negative cells are gated (green). ( C ) The proportions of GFP-positive HeLa (black columns) and A549 (white columns) target cells were quantified for each infection sample and are shown arranged by the Myc-PRMT6 plasmid cotransfected in the virus producer cells. Data are expressed relative to the respective empty vector sample (“None”), and columns represent the means and standard deviations of two independent infections using independent virus stocks.

Article Snippet: Endogenous PRMT6 was detected with rabbit anti-PRMT6 antibody (LifeSpan Biosciences).

Techniques: Produced, Infection, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Western Blot, Luciferase, Flow Cytometry

Journal: Virology Journal

Article Title: Overexpression of PRMT6 does not suppress HIV-1 Tat transactivation in cells naturally lacking PRMT6

doi: 10.1186/1743-422X-10-207

Figure Lengend Snippet: Oligonucleotide sequences of inverse PCR primers used to construct Myc PRMT6 and Tat-FLAG mutants

Article Snippet: Endogenous PRMT6 was detected with rabbit anti-PRMT6 antibody (LifeSpan Biosciences).

Techniques: Inverse PCR, Construct, Mutagenesis