Journal: PLoS ONE

Article Title: Fused toes homolog, a potential molecular regulator of human papillomavirus type 16 E6 and E7 oncoproteins in cervical cancer

doi: 10.1371/journal.pone.0266532

Figure Lengend Snippet: Expression of HPV16 E6/E7 and FTS in the cell lines (A). The expression levels of E6 and E7 in the control and FTS silenced group (B). The tumor suppressor proteins p53 and pRb were determined by western blot in the control and FTS silenced group (C). Actin was used as a loading control. The bar graphs adjacent to each blot represents mean ± standard error of the mean (SEM) of densitometric analyses performed on the blots obtained from three independent experiments. The data has been normalized to control. Differences between the groups were calculated by unpaired, parametric, two-tailed T-tests. p-values of statistically significant differences are shown. * p <0.05; ** p < 0.005; *** p < 0.001.

Article Snippet: This was followed by overnight incubation at 4°C with anti-FTS (cat. no. sc-134343, Santa Cruz) and either anti-E6 (cat. no. sc-1584, Santa Cruz) or anti-E7 (cat. no. bs-10446R, Bioss, USA) antibodies.

Techniques: Expressing, Western Blot, Two Tailed Test

Journal: PLoS ONE

Article Title: Fused toes homolog, a potential molecular regulator of human papillomavirus type 16 E6 and E7 oncoproteins in cervical cancer

doi: 10.1371/journal.pone.0266532

Figure Lengend Snippet: The levels of tumor suppressor proteins p53, pRb, and cell survival marker Akt and pAKT were determined by western blot in the control and E6 (A) or E7 (B) silenced groups. Actin was used as a loading control. The bar graphs adjacent to each blot represents mean ± SEM of densitometric analyses performed on the blots obtained from three independent experiments. The data has been normalized to control. Differences between the groups were calculated by unpaired, parametric, two-tailed T-tests. p-values of statistically significant differences are shown. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p <0.0001.

Article Snippet: This was followed by overnight incubation at 4°C with anti-FTS (cat. no. sc-134343, Santa Cruz) and either anti-E6 (cat. no. sc-1584, Santa Cruz) or anti-E7 (cat. no. bs-10446R, Bioss, USA) antibodies.

Techniques: Marker, Western Blot, Two Tailed Test

Journal: PLoS ONE

Article Title: Fused toes homolog, a potential molecular regulator of human papillomavirus type 16 E6 and E7 oncoproteins in cervical cancer

doi: 10.1371/journal.pone.0266532

Figure Lengend Snippet: mRNA expression of HPV16 E6/E7 was evaluated in FTS intact/silenced group (A). Further, FTS mRNA levels were also verified in HPV16 E6/E7 intact/silenced group (B). GAPDH was used as positive control.

Article Snippet: This was followed by overnight incubation at 4°C with anti-FTS (cat. no. sc-134343, Santa Cruz) and either anti-E6 (cat. no. sc-1584, Santa Cruz) or anti-E7 (cat. no. bs-10446R, Bioss, USA) antibodies.

Techniques: Expressing, Positive Control

Journal: PLoS ONE

Article Title: Fused toes homolog, a potential molecular regulator of human papillomavirus type 16 E6 and E7 oncoproteins in cervical cancer

doi: 10.1371/journal.pone.0266532

Figure Lengend Snippet: Expression of FTS and E6 in CaSki cells (A), FTS and E7 in CaSki cells (B), FTS and E6 in SiHa cells (C), and FTS and E7 in SiHa cells (D). FTS was stained with Alexa Fluor 488 (green), while E6 or E7 was stained with Alexa Fluor 594 (red). The nuclei were counter-stained with DAPI. The Pearson’s coefficient for FTS co-localization with E6/E7 revealed that the co-localization ranged between 0.834–0.938.

Article Snippet: This was followed by overnight incubation at 4°C with anti-FTS (cat. no. sc-134343, Santa Cruz) and either anti-E6 (cat. no. sc-1584, Santa Cruz) or anti-E7 (cat. no. bs-10446R, Bioss, USA) antibodies.

Techniques: Expressing, Staining

Journal: PLoS ONE

Article Title: Fused toes homolog, a potential molecular regulator of human papillomavirus type 16 E6 and E7 oncoproteins in cervical cancer

doi: 10.1371/journal.pone.0266532

Figure Lengend Snippet: IP shows binding between FTS and E6/E7 in CaSki and SiHa cells (A), expression and co-localization of FTS (green color) and HPV16 E6 (brown color) in cervical cancer tissues (B), and expression and co-localization of FTS (green color) and HPV16 E7 (red color) in cervical cancer tissues (C). The co-localized area with maximum intensity is enclosed by a black box. The images of IHC shown are representative of 10 cervical cancer tissues. Black bar in (B) and (C) corresponds to 20 μm.

Article Snippet: This was followed by overnight incubation at 4°C with anti-FTS (cat. no. sc-134343, Santa Cruz) and either anti-E6 (cat. no. sc-1584, Santa Cruz) or anti-E7 (cat. no. bs-10446R, Bioss, USA) antibodies.

Techniques: Binding Assay, Expressing

Journal: PLoS ONE

Article Title: Fused toes homolog, a potential molecular regulator of human papillomavirus type 16 E6 and E7 oncoproteins in cervical cancer

doi: 10.1371/journal.pone.0266532

Figure Lengend Snippet: Docking conformation of the HPV16 E6/FTS (A) and E7/FTS (D), interacting residues of the HPV16 E6/FTS (B) and E7/FTS (E) protein-protein complex depicted in sticks representation, three intermolecular H-bonds present in the interface of the complex illustrated by yellow dotted lines, HPV16 E6/FTS (C), and E7/FTS (F). In all the cases, the HPV16 E6/E7 and FTS are represented in cyan and magenta colors, respectively.

Article Snippet: This was followed by overnight incubation at 4°C with anti-FTS (cat. no. sc-134343, Santa Cruz) and either anti-E6 (cat. no. sc-1584, Santa Cruz) or anti-E7 (cat. no. bs-10446R, Bioss, USA) antibodies.

Techniques:

Journal: PLoS ONE

Article Title: Attenuated Recombinant Influenza A Virus Expressing HPV16 E6 and E7 as a Novel Therapeutic Vaccine Approach

doi: 10.1371/journal.pone.0138722

Figure Lengend Snippet: Following generation of recombinant H1N1 or H3N2 16E6E7/16E6E7m influenza A viruses and passaging for at least five cycles, A whole viral RNA was isolated or B Vero cells were infected with 10 5 pfu of recombinant viruses for 12 hours and whole RNA isolated. Viral or cellular RNA was subjected to RT-PCR analysis amplifying either the whole delNS1-E6E7 ORF ( A ) to analyze stability, or the E6E7 transgene ( B ) to assess fusion gene expression. C,D Vero cells were infected for 12 hours in the presence ( D ) or absence ( C ) of proteasome inhibitor MG-132 with H1N1 or H3N2 recombinant or parental influenza viruses as indicated, and whole cell lysates were separated by 10% SDS-PAGE. Viral NP protein ( C ), FLAG (upper panel), HPV16 E6 (middle panel) and E7 expression (lower panel) were assessed using specific monoclonal antibodies ( D ).

Article Snippet: The antibodies used in these assays comprised monoclonal mouse anti(α)-influenza A NP blend clone A1/A3 (Millipore, 1:1,000 dilution); mouse α-Flag clone M2 (Sigma, 1:5,000); mouse α-HPV16 E7 clone ED17 (Santa Cruz, 1:200); mouse α-HPV16 E7 clone 8C9 (Invitrogen, 1:150); mouse α-HPV16 E6 clone 6F4 (Euromedex, 1:200); mouse α-HPV16 L1 Camvir-1 (BD Biosciences, 1:5,000); mouse α-pRb (1:500 dilution); polyclonal rabbit α-HPV16 L2 11–200 (described in [ ], 1:5,000) and rabbit α-GAPDH (Santa Cruz, 1:1.000); goat α-rabbit HRP and goat α-mouse HRP (both from Biorad,1:25,000).

Techniques: Recombinant, Passaging, Isolation, Infection, Reverse Transcription Polymerase Chain Reaction, Gene Expression, SDS Page, Expressing, Bioprocessing

Journal: PLoS ONE

Article Title: Attenuated Recombinant Influenza A Virus Expressing HPV16 E6 and E7 as a Novel Therapeutic Vaccine Approach

doi: 10.1371/journal.pone.0138722

Figure Lengend Snippet: A Sf-9 insect cells were infected with recombinant HPV16 L1/L2-E7 baculovirus and VLP purified by sucrose cushion and CsCl gradient centrifugation. Purified VLP, infected, and uninfected Sf-9 cell lysates were analyzed by SDS-PAGE and Coomassie staining. B Antigenicity of HPV16 L1/L2-E7 VLP was assessed by Western blot using antibodies to HPV16 L1, HPV16 L2, or HPV16 E7. Purified HPV16 L1 VLP and HPV16 L1/L2 VLP were used as controls. C HPV16 L1/L2-E7 VLP were negatively stained by uranyl acetate and visualized by TEM at 30,000 fold magnification.

Article Snippet: The antibodies used in these assays comprised monoclonal mouse anti(α)-influenza A NP blend clone A1/A3 (Millipore, 1:1,000 dilution); mouse α-Flag clone M2 (Sigma, 1:5,000); mouse α-HPV16 E7 clone ED17 (Santa Cruz, 1:200); mouse α-HPV16 E7 clone 8C9 (Invitrogen, 1:150); mouse α-HPV16 E6 clone 6F4 (Euromedex, 1:200); mouse α-HPV16 L1 Camvir-1 (BD Biosciences, 1:5,000); mouse α-pRb (1:500 dilution); polyclonal rabbit α-HPV16 L2 11–200 (described in [ ], 1:5,000) and rabbit α-GAPDH (Santa Cruz, 1:1.000); goat α-rabbit HRP and goat α-mouse HRP (both from Biorad,1:25,000).

Techniques: Infection, Recombinant, Purification, Gradient Centrifugation, SDS Page, Staining, Western Blot

Journal: PLoS ONE

Article Title: Attenuated Recombinant Influenza A Virus Expressing HPV16 E6 and E7 as a Novel Therapeutic Vaccine Approach

doi: 10.1371/journal.pone.0138722

Figure Lengend Snippet: A Schematic illustration of the experimental set up. B Groups (n = 8) of C57BL/6 female mice were vaccinated s.c. with PBS (mock), H1N1 16E6E7, H1N1 16E6E7m, parental virus (delNS1), or HPV16 L1/L2-E7 VLP, boosted 20 days later either with PBS, the corresponding H3N2 influenza serotype, or VLPs and challenged s.c. with 5 x 10 4 TC-1 cells 10 days later. Animals were monitored once per week. Mean tumor volumes ± SEM of individual animals of one representative experiment of two are shown. Statistical significances of differences recorded for immunized groups and mock treated animals, or immunized groups and parental virus treated animals, were calculated by 1-way ANOVA and subsequent Dunnett’s multiple comparison test. Days after TC-1 tumor inoculation are indicated.

Article Snippet: The antibodies used in these assays comprised monoclonal mouse anti(α)-influenza A NP blend clone A1/A3 (Millipore, 1:1,000 dilution); mouse α-Flag clone M2 (Sigma, 1:5,000); mouse α-HPV16 E7 clone ED17 (Santa Cruz, 1:200); mouse α-HPV16 E7 clone 8C9 (Invitrogen, 1:150); mouse α-HPV16 E6 clone 6F4 (Euromedex, 1:200); mouse α-HPV16 L1 Camvir-1 (BD Biosciences, 1:5,000); mouse α-pRb (1:500 dilution); polyclonal rabbit α-HPV16 L2 11–200 (described in [ ], 1:5,000) and rabbit α-GAPDH (Santa Cruz, 1:1.000); goat α-rabbit HRP and goat α-mouse HRP (both from Biorad,1:25,000).

Techniques: Virus, Comparison

Journal: PLoS ONE

Article Title: Attenuated Recombinant Influenza A Virus Expressing HPV16 E6 and E7 as a Novel Therapeutic Vaccine Approach

doi: 10.1371/journal.pone.0138722

Figure Lengend Snippet: A Schematic illustration of the experimental set up. B Groups (n = 8) of C57BL/6 female mice were challenged with 5 x 10 4 TC-1 cells on day 0 and tumor growth monitored. Mice were primed s.c. after palpable tumors had developed on day 12 with PBS (mock), H1N1 16E6E7, H1N1 16E6E7m, parental influenza virus (delNS1), or HPV16 L1/L2-E7 VLP, and boosted 10 days later as described. Animals were monitored once per week. Mean tumor volumes ± SEM of individual animals of one representative experiment of two are shown. Statistical significance of differences recorded for immunized groups and mock treated animals were calculated by 1-way ANOVA and subsequent Dunnett’s multiple comparison test. Days after TC-1 tumor inoculation are indicated. C Termination criteria comprised rapid weight loss, tumor diameters >16 mm, tumor ulceration, scrappy fur and diarrhea. Days indicate time after TC-1 tumor cell inoculation. One representative experiment of two is shown. Asterisks indicate p-values compared to mock (*** p<0.001, ** p<0.01).

Article Snippet: The antibodies used in these assays comprised monoclonal mouse anti(α)-influenza A NP blend clone A1/A3 (Millipore, 1:1,000 dilution); mouse α-Flag clone M2 (Sigma, 1:5,000); mouse α-HPV16 E7 clone ED17 (Santa Cruz, 1:200); mouse α-HPV16 E7 clone 8C9 (Invitrogen, 1:150); mouse α-HPV16 E6 clone 6F4 (Euromedex, 1:200); mouse α-HPV16 L1 Camvir-1 (BD Biosciences, 1:5,000); mouse α-pRb (1:500 dilution); polyclonal rabbit α-HPV16 L2 11–200 (described in [ ], 1:5,000) and rabbit α-GAPDH (Santa Cruz, 1:1.000); goat α-rabbit HRP and goat α-mouse HRP (both from Biorad,1:25,000).

Techniques: Virus, Comparison

Journal: PLoS ONE

Article Title: Attenuated Recombinant Influenza A Virus Expressing HPV16 E6 and E7 as a Novel Therapeutic Vaccine Approach

doi: 10.1371/journal.pone.0138722

Figure Lengend Snippet: A Schematic illustration of the experimental set up B Five groups of female C57BL/6 mice (8 per group) were inoculated with 5x10 4 TC-1 cells on day 0 and tumor growth monitored. After palpable tumors had developed, mice were primed i.t. on day 13 and 14 with PBS (mock), H1N1 16E6E7, H1N1 16E6E7m, parental influenza virus (delNS1), or HPV16 L1/L2-E7 VLP, and boosted either with PBS, the corresponding H3N2 influenza serotype, or VLPs 10 days later. Animals were monitored once per week and mean tumor volumes ± SEM of individual animals of one representative experiment of two are shown. Statistical significance of differences recorded for immunized groups and mock treated animals, or immunized groups and parental virus treated animals, were calculated by 1-way ANOVA and subsequent Dunnett’s multiple comparison test. Days after TC-1 tumor inoculation are indicated.

Article Snippet: The antibodies used in these assays comprised monoclonal mouse anti(α)-influenza A NP blend clone A1/A3 (Millipore, 1:1,000 dilution); mouse α-Flag clone M2 (Sigma, 1:5,000); mouse α-HPV16 E7 clone ED17 (Santa Cruz, 1:200); mouse α-HPV16 E7 clone 8C9 (Invitrogen, 1:150); mouse α-HPV16 E6 clone 6F4 (Euromedex, 1:200); mouse α-HPV16 L1 Camvir-1 (BD Biosciences, 1:5,000); mouse α-pRb (1:500 dilution); polyclonal rabbit α-HPV16 L2 11–200 (described in [ ], 1:5,000) and rabbit α-GAPDH (Santa Cruz, 1:1.000); goat α-rabbit HRP and goat α-mouse HRP (both from Biorad,1:25,000).

Techniques: Virus, Comparison