Journal: Cancer Science

Article Title: Roles of the PDZ ‐binding motif of HPV 16 E6 protein in oncogenic transformation of human cervical keratinocytes

doi: 10.1111/cas.13264

Figure Lengend Snippet: Establishment of HCKT ‐E7‐ HRAS G 12V expressing the wild type HPV 16E6 or its mutants defective for degradation of p53 and/or PDZ domain containing proteins. Expression of HPV 16E7, HRAS mutant, HRAS G 12V , and HPV 16E6 or its mutants was introduced to HCK 1T cells by retrovirus mediated transduction as described in Materials and Method. The levels of the wild type 16E6 and its mutant were examined by Western blots (a) and RT ‐ PCR (b). (a) The mouse monoclonal antibody for HPV 16E6 was raised against 16 amino acids of its N‐terminal region and therefore does not react with HPV 16E6 SAT which contains R8S/P9A/R10T substitution. While the wild type E6 and E6Δ151 which lacks its C‐terminal amino acid induced p53 degradation, E6 mutants with SAT substituions such as E6 SAT and E6 SAT Δ151 did not do so. The levels of PDZ domain containing proteins, such as Scribble ( SCRIB ), DLG 4, MAGI ‐1 ( MAGI ) and PAR 3 were decreased in wild‐type E6, but not E6Δ151 or E6 SAT Δ151 expressing cells. The level of HPV 16E7 and HRAS were not affected by the expression of E6. α‐tubulin was detected as a loading control. (b) mRNA levels of the wild type E6 and its mutants were comparable in those cells. 36B4 mRNA was also detected as an internal control. (c) The level of PTPN 13 was compared in indicated cells by Western blotting.

Article Snippet: All other antibodies were purchased as followings: monoclonal antibodies against HPV16E7 (clone 8C9; Invitrogen, Carlsbad, CA, USA), DLG4 (PSD95) (Upstate, K28/43), DLG1 (Santa Cruz, Dallas, TX, USA BD Transduction lab, clone 12) and p53 (Oncogene Science/EMD Millipore, Billerica, MA, USA; Ab6), p21 (Oncogene Science, WAF1 Ab1), GAPDH (Ambion, Austin, TX, USA; AM4300) and polyclonal antibodies against HRAS (Santa Cruz, sc‐520), PAR3 (Upstate 07‐330 and MBL, D235‐3), SCRIB (Becton Dickinson, Franklin Lakes, NJ, USA; C‐20), MAGI1 (Sigma, M‐5691), α‐tubulin (Cell Signaling Technologies, Danvers, MA, USA; 2144S) and β‐actin (Santa Cruz, sc‐1616).

Techniques: Expressing, Mutagenesis, Transduction, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Cancer Science

Article Title: Roles of the PDZ ‐binding motif of HPV 16 E6 protein in oncogenic transformation of human cervical keratinocytes

doi: 10.1111/cas.13264

Figure Lengend Snippet: Tumorigenic potentials of HCK 1T‐E7‐ HRAS G 12V expressing the wild type or E6 mutants defective for inducing degradation of p53 and/or PDZ domain containing proteins. The ability of E6 mutants to promote cell proliferation was compared to the wild type by examining growth curve (a) and clonogenic potential (b and c). HCK 1T‐E7‐ HRAS G 12V cells transduced with retrovirus containing an empty vector (vector) or expressing the wild type or mutants of E6 were seeded at 2× 10 4 cells/well in 12 well plates at day 0 and cell number in each well was counted at indicated time points. ** indicates P ‐value < 0.05 compared to the wild type E6 expressing cells. (b, c) The cells were seeded on 35‐mm dishes under sparse conditions. After cultivating for 2 weeks, the cells were stained with Giemsa's dye, and number of colonies was counted. The photographs are representative dishes (b), and the graph illustrates means + SD s (c). Anchorage independent growth of HCK 1T‐E7‐ HRAS G 12V expressing wild type or mutants of E6 was examined (d, e). The cells were seeded onto soft agar at 5 × 10 4 and cultivated for 4 weeks. The representative images are shown (d). Colonies whose size was >50 μm in diameter were counted and the total number of colonies in a 16 mm 2 area was compared (e). Tumor promoting potentials of the E6 mutants were compared to the wild type by mouse xenografts. 1 × 10 6 cells mixed with Matrigel were subcutaneously injected into nude mice. Mice were sacrificed at 40 days after the injection and tumor weights were compared (f). P ‐value was evaluated by student's t ‐test.

Article Snippet: All other antibodies were purchased as followings: monoclonal antibodies against HPV16E7 (clone 8C9; Invitrogen, Carlsbad, CA, USA), DLG4 (PSD95) (Upstate, K28/43), DLG1 (Santa Cruz, Dallas, TX, USA BD Transduction lab, clone 12) and p53 (Oncogene Science/EMD Millipore, Billerica, MA, USA; Ab6), p21 (Oncogene Science, WAF1 Ab1), GAPDH (Ambion, Austin, TX, USA; AM4300) and polyclonal antibodies against HRAS (Santa Cruz, sc‐520), PAR3 (Upstate 07‐330 and MBL, D235‐3), SCRIB (Becton Dickinson, Franklin Lakes, NJ, USA; C‐20), MAGI1 (Sigma, M‐5691), α‐tubulin (Cell Signaling Technologies, Danvers, MA, USA; 2144S) and β‐actin (Santa Cruz, sc‐1616).

Techniques: Expressing, Transduction, Plasmid Preparation, Staining, Injection

Journal: Cancer Science

Article Title: Roles of the PDZ ‐binding motif of HPV 16 E6 protein in oncogenic transformation of human cervical keratinocytes

doi: 10.1111/cas.13264

Figure Lengend Snippet: The protein levels of PDZ containing proteins in cervical cancer cell lines. (a) The levels of PDZ containing proteins in HPV positive‐cervical cancer cell lines were compared to that in HPV negative‐cervical cancer cell lines or normal human keratinocytes by Western blot analysis. HaCaT cells are spontaneously immortalized human foreskin keratinocytes. (b) The effect of sh RNA to E6 AP on the level of PAR 3 in cervical cancer cells were examined by Western blot analysis. HeLa, SiHa and C33a cells with E6 AP sh RNA was generated as described previously. p53 was included as a positive control because an E6 and E6 AP complex was known to induce degradation of p53. α‐tubulin was also detected as a loading control. (c) The ability of E6 to induce reduction of PAR 3 level was compared between high and low risk HPV s. The expression E6 of high risk HPV s shown in red ( HPV 16, 18, 26, 30, 31, 33, 35, 39, 45, 52, 56, 58 and 59 or low‐risk HPV s shown in blue ( HPV 6, 11, 43 and 54) was introduced to HCK 1T by retrovirus mediated transduction. A retrovirus carrying empty vector was used as a control (vector). The levels of p53, MAGI ‐1 and PAR 3 were examined by Western blot analysis.

Article Snippet: All other antibodies were purchased as followings: monoclonal antibodies against HPV16E7 (clone 8C9; Invitrogen, Carlsbad, CA, USA), DLG4 (PSD95) (Upstate, K28/43), DLG1 (Santa Cruz, Dallas, TX, USA BD Transduction lab, clone 12) and p53 (Oncogene Science/EMD Millipore, Billerica, MA, USA; Ab6), p21 (Oncogene Science, WAF1 Ab1), GAPDH (Ambion, Austin, TX, USA; AM4300) and polyclonal antibodies against HRAS (Santa Cruz, sc‐520), PAR3 (Upstate 07‐330 and MBL, D235‐3), SCRIB (Becton Dickinson, Franklin Lakes, NJ, USA; C‐20), MAGI1 (Sigma, M‐5691), α‐tubulin (Cell Signaling Technologies, Danvers, MA, USA; 2144S) and β‐actin (Santa Cruz, sc‐1616).

Techniques: Western Blot, Generated, Positive Control, Expressing, Transduction, Plasmid Preparation

Journal: Cell

Article Title: Oligopeptide Signaling through Tb GPR89 Drives Trypanosome Quorum Sensing

doi: 10.1016/j.cell.2018.10.041

Figure Lengend Snippet: Oligopeptidase Expression In Vivo Drives Stumpy Formation, Related to Figure 7 Growth of parasites induced to express BIPN- Tb PGP, Tb PGP or Tb POP in vivo . In each case the parasitemias are shown of equivalent numbers of parasites inoculated to initiate the infection, doxycycline being provided to the mice from day 1 of infection. In all cases there is reduced growth upon peptidase expression, this being more pronounced and consistent for BIPN- Tb PGP than Tb PGP. Tb POP was only analyzed without a BIPN leader, because the protein is naturally secreted ( Figure S5 C). Each growth profile represents analysis of duplicate or triplicate infections for each condition. Error bars = SEM. The western blot demonstrates inducible Tb POP expression, with the expression of the stumpy marker PAD1 being present on the high density parasites not induced to express Tb POP and the low density parasites induced to express Tb POP. EF1 alpha provides a loading control. M; mouse.

Article Snippet: Primary antibody dilutions were prepared in 1% BSA/TBS and the membrane was incubated overnight. αGPR89 antibody was used at 1:1000, αBB2 antibody ( ) was used at 1:20 to detect the TY-tagged Tb GPR89, αPAD1 antibody ( ) was used at 1:1000 and αEF1 (elongation factor 1-alpha, Merck Millipore 05-235) was used for loading controls at 1:7000.

Techniques: Expressing, In Vivo, Infection, Western Blot, Marker