pkm2 expression vector  (Addgene inc)

Journal: Molecular Therapy Oncolytics

Article Title: Scutellarin resensitizes oxaliplatin-resistant colorectal cancer cells to oxaliplatin treatment through inhibition of PKM2

doi: 10.1016/j.omto.2021.03.010

Figure Lengend Snippet: Expression of PKM2 determines glycometabolism rate and sensitivity to oxaliplatin in SW480, OR-SW480, HT29, and OR-HT29 cells (A) Expression of PKM2 in SW480, OR-SW480, HT29, and OR-HT29 was evaluated by quantitative real-time PCR and western blot analysis. ∗p < 0.05. (B) Transfection efficiency of PKM2 siRNA (50 pmol/mL) and PKM2 plasmid (2 μg/mL) in SW480, OR-SW480, HT29, and OR-HT29 cells. (C) Glucose, lactate, and ATP assays were performed to evaluate the glycometabolism of SW480 and HT29 cells after treatment with oxaliplatin (1 μM) and PKM2 plasmid (2 μg/mL). ∗p < 0.05 versus empty plasmid group. (D) Glucose, lactate, and ATP assays were performed to evaluate the glycometabolism of OR-SW480 and OR-HT29 cells after treatment with oxaliplatin (10 μM) and PKM2 siRNA (50 pmol/mL). ∗p < 0.05 versus NCO group. # p < 0.05 versus Oxaliplatin + NCO group. (E) Effect of PKM2 plasmid (2 μg/mL) on inducing the oxaliplatin resistance in SW480 and HT29. ∗p < 0.05 versus Oxaliplatin + empty plasmid group. (F) Effect of PKM2 siRNA (50 pmol/mL) on reversing the oxaliplatin resistance in OR-SW480 and OR-HT29. ∗p < 0.05 versus oxaliplatin + NCO group.

Article Snippet: For enforced expression of PKM2, PKM2 expression vector was conducted by cloning the open reading frame of the PKM2 gene into the pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Plasmid Preparation

Journal: Molecular Therapy Oncolytics

Article Title: Scutellarin resensitizes oxaliplatin-resistant colorectal cancer cells to oxaliplatin treatment through inhibition of PKM2

doi: 10.1016/j.omto.2021.03.010

Figure Lengend Snippet: Scutellarin decreases the oxaliplatin resistance through inhibition of PKM2 in OR-SW480 and OR-HT29 cells (A) Effect of scutellarin (2 μM), oxaliplatin (10 μM), and PKM2 plasmid (2 μg/mL) on changing the expression of PKM2 in OR-SW480 and OR-HT29 cells. (B) Effect of PKM2 plasmid (2 μg/mL) on protecting the OR-SW480 and OR-HT29 cells from the cytotoxicity of co-treatment with oxaliplatin (10 μM) and scutellarin (2 μM). ∗p < 0.05 versus oxaliplatin + empty plasmid group. # p < 0.05 versus oxaliplatin + scutellarin + empty plasmid group. (C) PKM2 plasmid (2 μg/mL) increased the glycometabolism rate in scutellarin-treated (2 μM) OR-SW480 and OR-HT29 cells. ∗p < 0.05 versus empty plasmid group. # p < 0.05 versus oxaliplatin + empty plasmid group. & p < 0.05 versus oxaliplatin + scutellarin + empty plasmid group.

Article Snippet: For enforced expression of PKM2, PKM2 expression vector was conducted by cloning the open reading frame of the PKM2 gene into the pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA, USA).

Techniques: Inhibition, Plasmid Preparation, Expressing

Journal: Molecular Therapy Oncolytics

Article Title: Scutellarin resensitizes oxaliplatin-resistant colorectal cancer cells to oxaliplatin treatment through inhibition of PKM2

doi: 10.1016/j.omto.2021.03.010

Figure Lengend Snippet: Promotion of scutellarin on oxaliplatin-induced mitochondrial apoptosis pathway (A) Mitochondrial membrane potential (Δϕ) of OR-SW480 and OR-HT29 cells treated with oxaliplatin (10 μM), scutellarin (2 μM), and PKM2 plasmid (2 μg/mL) was measured by flow cytometry. (B) Release of cytochrome c and AIF was evaluated by western blot analysis after removal of mitochondria from cytosol of OR-SW480 and OR-HT29 cells treated with oxaliplatin (10 μM), scutellarin (2 μM), and PKM2 plasmid (2 μg/mL). (C) Western blot analysis was performed to detect the cleavage of caspase-9 and caspase-3 in OR-SW480 and OR-HT29 cells treated with oxaliplatin (10 μM), scutellarin (2 μM), and PKM2 plasmid (2 μg/mL). (D) Flow cytometry analysis was performed to detect the apoptotic rate of OR-SW480 and OR-HT29 cells treated with oxaliplatin (10 μM), scutellarin (2 μM), and PKM2 plasmid (2 μg/mL). ∗p < 0.05 versus empty plasmid group. # p < 0.05 versus oxaliplatin + empty plasmid group. & p < 0.05 versus oxaliplatin + scutellarin + empty plasmid group.

Article Snippet: For enforced expression of PKM2, PKM2 expression vector was conducted by cloning the open reading frame of the PKM2 gene into the pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA, USA).

Techniques: Plasmid Preparation, Flow Cytometry, Western Blot

Journal: Molecular Therapy Oncolytics

Article Title: Scutellarin resensitizes oxaliplatin-resistant colorectal cancer cells to oxaliplatin treatment through inhibition of PKM2

doi: 10.1016/j.omto.2021.03.010

Figure Lengend Snippet: Scutellarin sensitizes oxaliplatin-resistant CRC cells to oxaliplatin treatment in vivo (A) Tumor growth of mice bearing OR-SW480 cells after treatment with oxaliplatin (10 mg/kg) and scutellarin (10 mg/kg) twice a week. (B) Resected tumors from mice in each group. (C) Western blot assay was performed to detect the cleavage of caspase-9 and caspase-3 in the resected tumors. (D) Western blot assay was performed to analyze the expression of PKM2 in the resected tumors. (E) Production of ATP in the purified tumor cells in each group. ∗p < 0.05 versus control group. # p < 0.05 versus oxaliplatin group.

Article Snippet: For enforced expression of PKM2, PKM2 expression vector was conducted by cloning the open reading frame of the PKM2 gene into the pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA, USA).

Techniques: In Vivo, Western Blot, Expressing, Purification

Journal: Molecular Therapy Oncolytics

Article Title: Scutellarin resensitizes oxaliplatin-resistant colorectal cancer cells to oxaliplatin treatment through inhibition of PKM2

doi: 10.1016/j.omto.2021.03.010

Figure Lengend Snippet: Schema of the predicted mechanisms implicated in OR-SW480 cells response to oxaliplatin Scutellarin inhibits PKM2 expression and thus reduces the glycometabolism rate and the production of ATP. The lower level of ATP facilitates the oxaliplatin-induced mitochondrial dysfunction, as determined by a decrease in Δϕ. As a result, cytochrome c and AIF are released from the mitochondria into the cytosol. Subsequently, these apoptotic inducers activate the effector caspases and cause the final occurrence of apoptosis.

Article Snippet: For enforced expression of PKM2, PKM2 expression vector was conducted by cloning the open reading frame of the PKM2 gene into the pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA, USA).

Techniques: Expressing

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

Journal: Nature

Article Title: The metabolic function of cyclin D3-CDK6 kinase in cancer cell survival

doi: 10.1038/nature22797

Figure Lengend Snippet: a, Enrichment of GO terms among CDK6-interactors identified in all T-ALL cell lines. p-values, Benjamini-Hochberg-test. b, In vitro kinase reactions using immunoprecipitated endogenous CDK6 and recombinant PFKP or PKM2 ±palbociclib (PALBO). 32P-PFKP/PKM2 denotes phosphorylated proteins, IB, immunoblotting. c, Phosphorylation of PFKP and PKM2 (from Extended Data Fig. 2e). d, PFKP and PKM2 activity in cells transfected with empty vector (Vec), D3/CDK6, or kinase-dead CDK6 (D3/CDK6-KD). e, PFKP and PKM2 activity after palbociclib-treatment. Data are mean ±s.d. *P<0.05; **P<0.01 (two-tailed t-test). b,c, representative experiments, out of 2 independent experiments (b), or 3 independent experiments (c, error bars from technical replicates). d,e, n=3 biological replicates. See Supplementary Fig. 1 for gel source data.

Article Snippet: PKM2 expressing vector (pWZL Neo Myr Flag PKM2) was also from Addgene.

Techniques: In Vitro, Immunoprecipitation, Recombinant, Western Blot, Phospho-proteomics, Activity Assay, Transfection, Plasmid Preparation, Two Tailed Test

Journal: Nature

Article Title: The metabolic function of cyclin D3-CDK6 kinase in cancer cell survival

doi: 10.1038/nature22797

Figure Lengend Snippet: Flow of glucose-derived carbon into PPP (a), and serine pathway (b) following D3-CDK6 inhibition in T-ALL KOPTK1 cells expressing wild-type PFKP and PKM2 (KOPTK1-WT), or PFKP-S679E and PKM2-S37E mutants (KOPTK1-EE). Levels of NADPH (c), GSH (d), ROS (e) in T-ALL cell lines upon palbociclib-treatment. f, Apoptosis of cells treated with palbociclib and NAC. g, Apoptosis in KOPTK1-WT and KOPTK1-EE cells upon palbociclib-treatment, or following knockdown of CDK6 (h). i,j, In vivo apoptosis of T-ALL cells (in peripheral blood and bone marrow, gated on human CD45+cells) in mice xenografted with KOPTK1-WT or KOPTK1-EE cells. Data are mean ±s.d. *P<0.05; **P<0.01, ***P<0.001 (two-tailed t-test). a,b, n=4, c-h, n=3, i,j, n=5 biological replicates. See Source data for Fig. 2 for T-ALL xenograft experiments.

Article Snippet: PKM2 expressing vector (pWZL Neo Myr Flag PKM2) was also from Addgene.

Techniques: Derivative Assay, Inhibition, Expressing, Knockdown, In Vivo, Two Tailed Test

Journal: Nature

Article Title: The metabolic function of cyclin D3-CDK6 kinase in cancer cell survival

doi: 10.1038/nature22797

Figure Lengend Snippet: a,b, Phosphorylation of PFKP and PKM2 (from Extended Data Fig. 9i), PFKP and PKM2 activity (c,d), levels of NADPH (e), GSH (f), ROS (g) in palbociclib-treated D3/CDK6-high cells. h, Apoptosis of D3/CDK6-high EBC1 cells expressing wild-type PFKP and PKM2 (EBC1-WT), or PFKP-S679E and PKM2-S37E mutants (EBC1-EE). Data are mean ±s.d. *P<0.05; **P<0.01, ***P<0.001 (two-tailed t-test). a,b, representative experiments (out of 3 independent experiments, error bars from technical replicates), c-h, n=3 biological replicates.

Article Snippet: PKM2 expressing vector (pWZL Neo Myr Flag PKM2) was also from Addgene.

Techniques: Phospho-proteomics, Activity Assay, Expressing, Two Tailed Test

Journal: Nature

Article Title: The metabolic function of cyclin D3-CDK6 kinase in cancer cell survival

doi: 10.1038/nature22797

Figure Lengend Snippet: PFKP and PKM2 phosphorylation (a,b, from Extended Data Fig. 10i, j), PFKP and PKM2 activity (c,d), GSH (e) and ROS (f) levels in D3/CDK6-high (regressing) and D3/CDK6-low (non-regressing) tumors from ribociclib-treated mice. Data are mean ±s.d. P<0.05; **P<0.01, ***P<0.001 (two-tailed t-test). n=3 biological replicates. See Source data for Fig. 5 for PDX drug treatment experiments.

Article Snippet: PKM2 expressing vector (pWZL Neo Myr Flag PKM2) was also from Addgene.

Techniques: Phospho-proteomics, Activity Assay, Two Tailed Test