Fig 1: Piwil2 knockdown affects cervical cancer cell line proliferation, invasion, and in vivo tumorigenicitya. HeLa, SiHa, and CaSki cells were stably transfected with control shRNA or Piwil2 shRNA, and cell viability was measured daily. b. Numbers of invading cells in clones stably transfected with control shRNA and Piwil2 shRNA. c. Equal amounts of lysates from cancer cell lines stably transfected with control shRNA or Piwil2 shRNA were separated by SDS-PAGE, and proteins were analyzed by western blotting with specific antibodies against Piwil2 and molecules regulating cell proliferation. d. Tumor growth over time was measured after the subcutaneous injection of 5×106 of SiHa cells stably transfected with shPiwil2 control shRNA and Piwil2 shRNA. Tumor volume was monitored by caliper measurements twice weekly, and tumor weight was measured after sacrifice at the end of the experiment. The data are presented as the mean±SD. *P < 0.05 and ** P < 0.01 by Student's t-test.
Fig 2: Model of cell reprogramming and tumor-initiating cell formation secondary to Piwil2 induction
Fig 3: Piwil2 initiates tumorigenicity of HaCaT cellsa. Approximately 5×106 HaCaT cells transfected with Piwil2 or Vector were injected subcutaneously into the oxters of nude mice. Four weeks after injection, tumorigenesis in nude mice was observed. b. Tumor volume was monitored by caliper measurements twice a week, and tumor weight was measured after sacrifice at the end of the experiment. The data are presented as the mean±SD. ** P < 0.01 by Student's t-test.
Fig 4: Characteristic Piwil2 expression in cervical cancer and its precursor stagesa. Piwil2 expression detected by IHC in CIN lesion and cervical cancer tissue. Scale bar, 100 μm. b. Immunoreactive score of Piwil2 staining in CIN lesions and cervical cancer tissue. c. Western blot analysis of Piwil2 expression in CIN lesions and cervical cancer tissue. GAPDH expression is presented as a loading control. d. Western blot analysis of Piwil2 expression in cervical cancer cell lines.
Fig 5: Piwil2 overexpression induces HaCaT cell malignant transformationa. HaCaT cells were transfected with lentivirus containing Piwil2 or lentiviral vector, and cell numbers were plotted daily. b.-c. Colony formation numbers and invading HaCaT cells infected with lentivirus containing Piwil2 or lentiviral vector. d. Proteins were analyzed by western blotting for c-Myc, E-cadherin, and molecules regulating cell proliferation and apoptosis. e.-f. The expression of EMT markers was determined by qRT-PCR and western blotting in HaCaT cells infected with lentivirus containing Piwil2 or lentiviral vector. The data are presented as the mean±SD. *P < 0.05 and ** P < 0.01 by Student's t-test.
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