Journal: Cancer Medicine

Article Title: Nocardia rubra cell‐wall skeleton influences the development of cervical carcinoma by promoting the antitumor effect of macrophages and dendritic cells

doi: 10.1002/cam4.4526

Figure Lengend Snippet: Forward and reverse primers

Article Snippet: The following primary antibodies were bounded with the proteins: mouse monoclonal anti‑HPV16+18‐E6 (1:1000, ab70, abcam, USA), rabbit polyclonal: monoclonal anti‐HPV18‐E7 (1:1000, ab100953, abcam, USA), anti‐HPV16‐E7 (1:1000, P03129, Bioss, China), anti‐p53 (1:10000, ab32389, abcam, USA), PD‐L1 (1:2000, ab205921, abcam, USA), anti‐caspase‐3 (1:5000, ab32351, abcam, USA), anti‐caspase‐8 (1:500, ab32397, abcam, USA), anti‐caspase‐9 (1:2000, ab32539, abcam, USA), anti‐BAX (1:2000, ab32503, abcam, USA), anti‐Bcl‐2 (1:1000, ab32124, abcam, USA), anti‐β‐catenin (1:1000, ab68183, abcam, USA), anti‐E‐cadherin (1:2000, ab40772, abcam, USA), anti‐N‐cadherin (1:2000, ab207608, abcam, USA), and anti‐TNFR1 (1:1000, ab259817, abcam, USA).

Techniques: Sequencing

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