Journal: Viruses

Article Title: Non-Metabolic Functions of PKM2 Contribute to Cervical Cancer Cell Proliferation Induced by the HPV16 E7 Oncoprotein

doi: 10.3390/v13030433

Figure Lengend Snippet: PKM2 is upregulated and has a prognostic value in cervical cancer. ( A ) TPM values of PKM2 mRNA in cervical cancer tissues ( n = 304) and normal cervical tissues ( n = 11) are shown in the log scale. * p = 1.3 × 10 −6 (Welch’s unequal variances t -test). ( B ) Cervical cancer tissues shown in A were grouped based on the HPV status. The level of PKM2 mRNA was 2.1-fold higher in HPV + cervical cancer ( n = 169) than HPV – cervical cancer ( n = 9). * p = 0.05 (Welch’s unequal variances t -test). ( C ) Overall survival was worse in cervical cancer patients with high levels of PKM2 (red, n = 76) than those with low levels of PKM2 (blue, n = 75).

Article Snippet: Retroviral pGFP-V-RS vectors expressing PKM2 shRNAs (Cat. No. TG302462) and scrambled shRNA (Cat. No. TR30013) were purchased from OriGene (Rockville, MD, USA), and the PKM2 shRNA clone B was used for experiments.

Techniques:

Journal: Viruses

Article Title: Non-Metabolic Functions of PKM2 Contribute to Cervical Cancer Cell Proliferation Induced by the HPV16 E7 Oncoprotein

doi: 10.3390/v13030433

Figure Lengend Snippet: HPV16 E7 upregulates and interacts with PKM2 in cervical cancer cells. ( A ) HPV16 E7 interacts with PKM2 in SiHa cells. SiHa cell extracts (0.5 mg) were subject to co-immunoprecipitation using an anti-HPV16 E7 antibody. Normal IgG was used as a negative control. The input lane represents 50 μg of cell extracts. ( B ) GST proteins fused to HPV18 E7 (GST-18E7) and HPV45 E7 (GST-45E7) were incubated with 293T cell extracts overexpressing HA-tagged PKM2 (HA-PKM2). GST was used as a negative control. PKM2 was detected by western blot using an anti-HA antibody. GST fusion proteins were visualized by Coomassie blue staining. Intervening lanes were deleted and indicated by vertical lines. ( C ) PKM2 levels were higher in HPV + than in HPV − cervical cancer cells. Cell extracts were subject to western blot. Intervening lanes were deleted and indicated by vertical lines. ( D ) The levels of PKM2 mRNA was higher in SiHa cells than C33A cells. Total RNA was subject to semi-quantitative RT-PCR. The number of PCR cycle was 25 for PKM2 and 22 for GAPDH . ( E ) HPV16 E7 increased the level of PKM2. Cells were transfected with an empty (mock) or HPV16 E7-expressing plasmid. Cell extracts were subject to western blot. Actin was used as a loading control.

Article Snippet: Retroviral pGFP-V-RS vectors expressing PKM2 shRNAs (Cat. No. TG302462) and scrambled shRNA (Cat. No. TR30013) were purchased from OriGene (Rockville, MD, USA), and the PKM2 shRNA clone B was used for experiments.

Techniques: Immunoprecipitation, Negative Control, Incubation, Western Blot, Staining, Quantitative RT-PCR, Transfection, Expressing, Plasmid Preparation, Control

Journal: Viruses

Article Title: Non-Metabolic Functions of PKM2 Contribute to Cervical Cancer Cell Proliferation Induced by the HPV16 E7 Oncoprotein

doi: 10.3390/v13030433

Figure Lengend Snippet: HPV16 E7-induced proliferation of cervical cancer cells depend on PKM2. ( A ) C33A cells stably expressing HPV16 E7 or empty vector were generated using retroviral vectors. Cell extracts were analyzed with western blot. ( B ) Cells described in A were seeded in 24-well plates (20,000 cells/well) and counted after 5 days. Results from five independent experiments are shown as mean ± S.E.M. V, vector. * p = 0.005 (two-sided Student’s t -test). ( C ) C33A-E7 cells were transduced with retrovirus expressing scrambled (SC) shRNA or PKM2 shRNA. Total cell extracts were subject to western blot. ( D ) Cells described in C were subject to cell counting assay, as described in B. Results from three independent experiments are shown as mean ± S.E.M. * p = 0.01 (two-sided Student’s t -test).

Article Snippet: Retroviral pGFP-V-RS vectors expressing PKM2 shRNAs (Cat. No. TG302462) and scrambled shRNA (Cat. No. TR30013) were purchased from OriGene (Rockville, MD, USA), and the PKM2 shRNA clone B was used for experiments.

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Generated, Retroviral, Western Blot, Transduction, shRNA, Cell Counting

Journal: Viruses

Article Title: Non-Metabolic Functions of PKM2 Contribute to Cervical Cancer Cell Proliferation Induced by the HPV16 E7 Oncoprotein

doi: 10.3390/v13030433

Figure Lengend Snippet: PKM2 is required for proliferation of cervical cancer cells. ( A ) SiHa cells were transiently transfected with scrambled (SC) shRNA or PKM2 shRNA vector. Total cell extracts were analyzed with western blot. ( B ) SiHa cells were transiently transfected as described in A and photographed 5 days later. Note that cells transfected with an PKM2 shRNA plasmid were sub-confluent. Scale bar, 100 µm. ( C ) SiHa cells were transiently transfected as described in A and counted 5 days later. Data are presented as mean ± S.E.M. ( n = 3). * p = 0.01 (two-sided Student’s t -test).

Article Snippet: Retroviral pGFP-V-RS vectors expressing PKM2 shRNAs (Cat. No. TG302462) and scrambled shRNA (Cat. No. TR30013) were purchased from OriGene (Rockville, MD, USA), and the PKM2 shRNA clone B was used for experiments.

Techniques: Transfection, shRNA, Plasmid Preparation, Western Blot

Journal: Viruses

Article Title: Non-Metabolic Functions of PKM2 Contribute to Cervical Cancer Cell Proliferation Induced by the HPV16 E7 Oncoprotein

doi: 10.3390/v13030433

Figure Lengend Snippet: ML265 decreases the phosphorylation of PKM2 at the Y105 position. ( A ) SiHa cells were treated with ML265 (40 µm) or vehicle (veh) for 24 h and then treated with paraformaldehyde for cross-linking. Total cell extracts were analyzed with western blot. GAPDH was used as a loading control. Note that ML265 decreased monomer and increased tetramer. ( B ) SiHa cells were treated with vehicle and ML265 for 24 h. Cells were nearly confluent at the endpoint. The cytoplasmic fraction (C) and the nuclear fraction (N) were analyzed with western blot. GAPDH and lamin A/C were used as a cytoplasmic and nuclear fraction marker, respectively. ( C ) SiHa cells were treated with ML265 for 24 h. Total cell extracts were subject to western blot analysis. ( D ) Total cell extracts from C33A-vector and C33A-E7 cells described in A were analyzed with western blot. Note that both pY105-PKM2 and total PKM2 were increased in C33A-E7 cells compared to the control cells.

Article Snippet: Retroviral pGFP-V-RS vectors expressing PKM2 shRNAs (Cat. No. TG302462) and scrambled shRNA (Cat. No. TR30013) were purchased from OriGene (Rockville, MD, USA), and the PKM2 shRNA clone B was used for experiments.

Techniques: Phospho-proteomics, Western Blot, Control, Marker, Plasmid Preparation