Journal: Journal of Virology

Article Title: Uncovering the Role of the E1 Protein in Different Stages of Human Papillomavirus 18 Genome Replication

doi: 10.1128/JVI.00674-20

Figure Lengend Snippet: The replication efficiency of the HPV18-E1HA-Nluc-E2Flag genome is similar to that of the WT HPV18. (A) Map of the HPV18-E1HA-Nluc-E2Flag genome. Red indicates the position of the tags in the E1 and E2 ORFs. The sequence of Nluc is immediately after 72 nucleotides of the WT E2 ORF, which overlaps with the 3′ end of the E1-HA ORF. The full-length E2 ORF containing the Flag tag sequence follows immediately after the sequence encoding the self-processing 2A peptide situated in the 3′ end of the E2-Nluc ORF (not shown). (B) Transient replication of the HPV18 and HPV18-E1HA-Nluc-E2Flag genomes in the U2OS cells was analyzed using SB. Total DNA was treated with BglI and DpnI restriction enzymes. (C and D, left panel) SB signals corresponding to the replicated HPV18 (C) and HPV18-E1HA-Nluc-E2Flag genome (D) were quantified using ImageQuant software. The percent intensity was calculated for each sample relative to the signals obtained in the 2-day posttransfection cells. (D, right panel) The samples obtained from the cells transfected with HPV18-E1HA-Nluc-E2Flag were analyzed for Nluc activity, which was normalized by the activity of alkaline phosphatase and set as 100% for the 2-day-posttransfection cells (*, P < 0.05; **, P < 0.01; n = 3). (E) U2OS cells were transfected with the HPV18-E1HA-Nluc-E2Flag genome and incubated for 2, 3, and 4 days. Linear regression of the normalized Nluc activity and quantified SB signals was analyzed using GraphPad software. The data are expressed as percentages ± the standard deviations (SD) of the signals obtained in the cells incubated for 2 days (set as 100%).

Article Snippet: Human osteosarcoma cell line U2OS (ATCC no HTB-96) and its derivatives were propagated in normal growth medium (NGM) containing Iscove modified Dulbecco medium (Pan Biotech), 10% fetal calf serum, and 1% penicillin-streptomycin (Sigma-Aldrich).

Techniques: Sequencing, FLAG-tag, Software, Transfection, Activity Assay, Incubation

Journal: Journal of Virology

Article Title: Uncovering the Role of the E1 Protein in Different Stages of Human Papillomavirus 18 Genome Replication

doi: 10.1128/JVI.00674-20

Figure Lengend Snippet: E1 RNAi restrains the transient replication of the modified HPV18 genomes in the U2OS cells. (A) U2OS cells were transfected with either the HPV18 or HPV18-E1HA-Nluc-E2Flag genome in the presence of E1-specific or Neg. siRNAs. The cells were incubated for 4 days. The endogenous HA-tagged E1 and Flag-tagged E2 proteins were immunoprecipitated and analyzed using WB and anti-tag antibodies. E1 was also detected in E2 immunocomplexes. GAPDH was used as a control. (B) WB signals corresponding to the E1-HA and E2-Flag protein levels were quantified and expressed as percentages of the E1-HA and E2-Flag levels, respectively, obtained in the samples transfected with Neg. siRNA (indicated as 100%). The data are expressed as average means ± the SD (***, P < 0.001; n = 4). (C) The U2OS cells were transfected with the HPV18-E1HA-Nluc-E2Flag or HPV18 WT genome, incubated for 3 days, and treated with DMSO or 9 μM CX4945 for 12, 24, or 48 h. Endogenous E1 and E2 proteins were immunoprecipitated and analyzed using WB and tag-specific antibodies. (D) WB signals corresponding to the E1-HA and E2-Flag protein levels were quantified and expressed as percentages of the E1-HA and E2-Flag levels, respectively, obtained in the samples treated with DMSO (indicated as 100%). The data are expressed as average means ± the SD (*, P < 0.05; ***, P < 0.001; n = 4). (E) U2OS cells were cotransfected with the HPV18-E1HA-Nluc-E2Flag genome, E1-specific or Neg. siRNA, and the constructs encoding either E1 or E2 of HPV18. Cells were incubated for 2, 3, or 4 days and subjected to luciferase assay. The Nluc activity was normalized to the activity of AP. The data are shown as a percentage of the normalized Nluc activity obtained in the cells transfected with the HPV18-E1HA-Nluc-E2Flag genome, Neg. siRNA and empty vector and incubated for 2 days (set as 100%) (for statistical analysis, data of each sample was compared to the data of the respective control at the same time point [**, P < 0.01; ***, P < 0.001; n = 3]). (F) U2OS cells were transfected with the HPV18-E1HA genome, E1-specific or Neg. siRNA, and E2-encoding construct. Total DNA was isolated from 2-, 3-, and 4-day-posttransfection cells, treated with BglI and DpnI restriction endonucleases and subjected to SB. (G) Cell cycle profile of the U2OS cells cotransfected with the HPV18-E1HA genome and the siRNAs was analyzed using PI.

Article Snippet: Human osteosarcoma cell line U2OS (ATCC no HTB-96) and its derivatives were propagated in normal growth medium (NGM) containing Iscove modified Dulbecco medium (Pan Biotech), 10% fetal calf serum, and 1% penicillin-streptomycin (Sigma-Aldrich).

Techniques: Modification, Transfection, Incubation, Immunoprecipitation, Control, Construct, Luciferase, Activity Assay, Plasmid Preparation, Isolation

Journal: Journal of Virology

Article Title: Uncovering the Role of the E1 Protein in Different Stages of Human Papillomavirus 18 Genome Replication

doi: 10.1128/JVI.00674-20

Figure Lengend Snippet: E1 RNAi is responsible for the suppression of the HPV18 replication. (A) U2OS cells were transfected with the constructs expressing HPV18 E1-WT, E1-siRNA1-R, and E1-siRNA2-R transcripts. E1-siRNA1-R and E1-siRNA2-R ORFs contain silent mutations to generate resistance for E1 siRNA1 and siRNA2, respectively. Housekeeping GAPDH and overexpressed E1-HA proteins were analyzed using WB. (B) U2OS cells were cotransfected with the HPV18-Nluc genome and different E1-HA expression constructs or an empty vector in quadruplicates. The cells were incubated for 2, 3, and 4 days and subjected to a luciferase assay. The Nluc activity was normalized to the total protein amount and set as 100% in the control cells cotransfected with the genome and empty vector and incubated for 2 days. The data are shown as average means ± the SD (n = 2). (C) E1-siRNA1-R and E1-siRNA2-R constructs were cotransfected with the E1 siRNAs into U2OS cells. After 3 days of transfection, GAPDH and E1-HA proteins were analyzed using immunoblotting. (D) U2OS cells were cotransfected with the HPV18-Nluc genome, E1 siRNAs, and E1-siRNA1-R and E1-siRNA2-R expression constructs in quadruplicates; incubated for 2, 3, and 4 days; and analyzed using luciferase assay. Nluc activity was normalized to the total protein amount and set as 100% in the control cells cotransfected with the genome, empty vector and Neg. siRNA and incubated for 2 days. The data are shown as average percentages of the control ± the SD (**, P < 0.01; ***, P < 0.001; n = 3). (E) U2OS cells were transfected with the indicated HPV18 genomes and incubated for 2, 3, and 4 days. Total DNA was extracted, digested with BglI and DpnI restriction enzymes, and analyzed using SB. (F) U2OS cells were cotransfected with either HPV18-E1HA-R1 or HPV18-E1HA-R2 genomes and Neg. or E1 siRNAs. Cells were incubated for 3 days. E1-HA and GAPDH proteins were analyzed using WB. (G) U2OS cells were cotransfected with either HPV18-E1HA-R1 or HPV18-E1HA-R2 genomes, E1 siRNAs, and E1-siRNA1-R or E1-siRNA2-R expression constructs, if indicated. After 3 days of incubation, total DNA was extracted, treated with BglI and DpnI restriction endonucleases, and subjected to SB analysis.

Article Snippet: Human osteosarcoma cell line U2OS (ATCC no HTB-96) and its derivatives were propagated in normal growth medium (NGM) containing Iscove modified Dulbecco medium (Pan Biotech), 10% fetal calf serum, and 1% penicillin-streptomycin (Sigma-Aldrich).

Techniques: Transfection, Construct, Expressing, Plasmid Preparation, Incubation, Luciferase, Activity Assay, Control, Western Blot

Journal: Journal of Virology

Article Title: Uncovering the Role of the E1 Protein in Different Stages of Human Papillomavirus 18 Genome Replication

doi: 10.1128/JVI.00674-20

Figure Lengend Snippet: U2OS cells support stable replication of the modified HPV18 genomes. (A) U2OS-derived HPV18-E1HA+ and HPV18-Nluc+ cells containing the stably replicating HPV18-E1HA and HPV18-Nluc genomes, respectively, were cultured for approximately two months. Total DNA was isolated at passages p2 to p12, treated with the restriction enzyme BglI linearizing the indicated HPV18 genomes, and subjected to SB. (B) Prior to SB, the LMW DNA isolated from the nonconfluent HPV18-E1HA+ and HPV18-Nluc+ cells (left and right panels, respectively) was treated with restriction endonucleases for either linearizing (lin) or not cutting (nc) the indicated modified HPV18 genomes. lin1, BglI; lin2, BglII; lin3, SdaI; nc1, HindIII; nc2, SacI; nc3, ScaI. (C) After seeding, the HPV18-E1HA+ and HPV18-Nluc+ stable cell lines were continuously propagated for 2, 4, and 7 days, resulting in nonconfluent, subconfluent, and confluent cultures, respectively. The U2OS cells were transfected with the respective modified HPV18 genomes and cultured for the indicated periods of time. Prior to SB, the isolated extrachromosomal DNA was treated with the restriction enzymes for either linearizing (BglI) or not cutting (HindIII) the indicated HPV18 genomes and with DpnI in the case of the transiently replicating genomes. Numbers indicate different forms of the uncut modified HPV18 DNA detected in the LMW DNA pool isolated from the confluent stable cell lines. ccc, covalently closed circular DNA; lin, linear DNA; oc, open circular DNA. (D) SB signals corresponding to the bands numbered in panel C were quantified. The sum of the pixels obtained from each sample was set as 100%, and the intensity of each particular band was calculated relative to 100%. The data are presented as average means ± the SD of at least five different samples.

Article Snippet: Human osteosarcoma cell line U2OS (ATCC no HTB-96) and its derivatives were propagated in normal growth medium (NGM) containing Iscove modified Dulbecco medium (Pan Biotech), 10% fetal calf serum, and 1% penicillin-streptomycin (Sigma-Aldrich).

Techniques: Modification, Derivative Assay, Stable Transfection, Cell Culture, Isolation, Transfection

Journal: Journal of Virology

Article Title: Uncovering the Role of the E1 Protein in Different Stages of Human Papillomavirus 18 Genome Replication

doi: 10.1128/JVI.00674-20

Figure Lengend Snippet: The HPV18-E1HA+ and HPV18-Nluc+ cells carry head-to-tail concatemeric episomes that replicate via non-theta-type intermediates. (A) LMW DNA was extracted via Hirt lysis from the subconfluent HPV18-E1HA+ and HPV18-Nluc+ cells. Prior to SB, equal amounts of LMW DNA (20 μg for HPV18-E1HA+ cells and 15 μg for HPV18-Nluc+ cells) were treated with 1 μl of HindIII and the indicated amounts of BglI restriction endonucleases, which serve as a noncutter and a single cutter, respectively. Reaction mixtures were incubated at 37°C for 30 min. DNA molecules with sizes corresponding to single, dimeric, trimeric, and tetrameric linear forms are indicated as 1 lin, 2 lin, 3 lin, and 4 lin, respectively. ccc, covalently closed circular DNA; oc, open circular DNA. (B) LMW DNA was isolated from the nonconfluent HPV18-Nluc+ cells and was subjected to neutral-neutral 2D analysis, using 0.4% agarose in Tris-borate-EDTA (TBE) in the first dimension and 1.2% agarose plus ethidium bromide in TBE in the second dimension. Viral DNA was detected using SB. Different forms of viral episomes from monomeric to hexameric genomes were detected. ccc, covalently closed circular DNA; oc, open circular DNA. (C) LMW DNA was isolated from the U2OS cells transiently transfected with the HPV18-Nluc genome (left panel) and from the HPV18-Nluc+ cells (right panel). DNA was digested with BglI, which cuts the viral genomes once at the origin of replication, and subjected to neutral-neutral 2D analysis as in panel B. Both the theta type (arrowhead) and the non-theta type of replication intermediates (arrows) were observed in the transiently transfected cells, while the non-theta type of replication intermediates prevailed in the HPV18-Nluc+ cells. Asterisks depict the almost fully replicated HPV DNA.

Article Snippet: Human osteosarcoma cell line U2OS (ATCC no HTB-96) and its derivatives were propagated in normal growth medium (NGM) containing Iscove modified Dulbecco medium (Pan Biotech), 10% fetal calf serum, and 1% penicillin-streptomycin (Sigma-Aldrich).

Techniques: Lysis, Incubation, Isolation, Transfection

Journal: PLoS ONE

Article Title: MITF-Independent Pro-Survival Role of BRG1-Containing SWI/SNF Complex in Melanoma Cells

doi: 10.1371/journal.pone.0054110

Figure Lengend Snippet: A. Effect of BRG1 or MITF depletion on DNA content profile determined by flow cytometry in 501mel cells. Cells were analyzed 2 days after selection in 2.5 µg/ml puromycin (left), or combined (detached and adherent) cells were analyzed 5 days after selection (right). All cells killed by puromycin were removed shortly after antibiotic selection and only puromycin resistant cells were assayed. Right, Western blots verifying the knockdown efficiency for BRG1 and MITF were performed after 2 days followimg the selection. B. BRG1 or MITF depletion inhibits colony formation in 501mel cells. A427 cells (BRG1 and BRM negative) were not affected by sh-BRG1. Middle, the quantitation of cells on dishes shown on the left. Right, Western blots verifying the knockdown efficiency for BRG1 and MITF. C. BRM depletion inhibits cell growth in BRG1-negative SK-MEL-5 melanoma cells, but not control RPMI7951 melanoma cells, which are BRM and BRG1-positive but MITF-negative . Right, Western blots showing the efficient shRNA-mediated BRM downregulation. *, not statistically significant (P<0.01).

Article Snippet: A427 lung carcinoma cells (ATCC, Manassas, VA) were grown in the RPMI-1640 medium and SK-MEL-5 and MeWo melanoma cells (ATCC) were maintained in the E-MEM medium supplemented with FBS, pyruvate, and non-essential amino acids (Sigma).

Techniques: Flow Cytometry, Selection, Western Blot, Knockdown, Quantitation Assay, Control, shRNA

Journal: PLoS ONE

Article Title: MITF-Independent Pro-Survival Role of BRG1-Containing SWI/SNF Complex in Melanoma Cells

doi: 10.1371/journal.pone.0054110

Figure Lengend Snippet: Viability of each cell line is indicated in the histogram. The results are expressed as per cents of control shRNA-transduced cells. The cells were infected with appropriate lentivirus, replated two days later into 12-well plates, and viability was determined after next 3 days. The values are means of triplicates +-SE. One of the three independent experiments is shown. No decrease of viability was observed in BRM/BRG1-null A427 cells, used as a negative control. The knockdown of BRG1, BRM or common BRG1/BRM was efficient (see , , and , and in our previous article ).

Article Snippet: A427 lung carcinoma cells (ATCC, Manassas, VA) were grown in the RPMI-1640 medium and SK-MEL-5 and MeWo melanoma cells (ATCC) were maintained in the E-MEM medium supplemented with FBS, pyruvate, and non-essential amino acids (Sigma).

Techniques: Control, shRNA, Infection, Negative Control, Knockdown