Journal: Cell

Article Title: VEGAS as a Platform for Facile Directed Evolution in Mammalian Cells

doi: 10.1016/j.cell.2019.05.051

Figure Lengend Snippet: Key Resources Table

Article Snippet: The following day membranes were washed 4 × 15 min in TBST and probed for 1 hr with anti-rabbit IgG HRP (1:5000, Jackson ImmunoResearch, #711-035-152) for mVenus detection or anti-mouse IgG HRP (1:3000, Cell Signaling, #7076S) for FLAG detection.

Techniques: Recombinant, Magnetic Beads, Expressing, Sonication, Western Blot, Isolation, In Vitro, Transfection, Plasmid Preparation, Gel Extraction, RNA Binding Assay, Enzyme-linked Immunosorbent Assay, Modification

Journal: Advanced Science

Article Title: Pyruvate Facilitates FACT‐Mediated γ H2AX Loading to Chromatin and Promotes the Radiation Resistance of Glioblastoma

doi: 10.1002/advs.202104055

Figure Lengend Snippet: PKM2 is required for γ H2AX levels in chromatin, DNA repair, and tumor cell survival. Immunoblotting (IB) experiments were performed with the indicated antibodies. Data are representative of at least three independent experiments. A) U251 cells stably expressing a nontargeting shRNA or PKM2 shRNA were rescued with or without rPKM2. Cells were treated with etoposide (200 × 10 –6 m ) for indicated time. Cell viability was determined using Trypan blue staining. Data represent the mean ± SD of the viability of the cells from three independent experiments (two‐tailed Student's t‐test). P values for comparisons between shNT and shPKM2 are shown in blue; comparisons between shPKM2 and shPKM2+rPKM2 are shown in green. B) U251 cells stably expressing shNT or shPKM2 were treated with or without etoposide (500 × 10 –6 m ) for 3 h. The representative images of the comet assay in these cells were shown (left panel). Scatter dot plot (right panel) of the tail moments in the comet assay from shNT‐expressing cells ( n = 86) or shPKM2‐expressing cells ( n = 86) treated with etoposide. Data represent mean ± SD of the tail moments (Mann Whitney test). Data are representative of three independent experiments. C,D) U251 cells stably expressing shNT or shPKM2 were treated with or without etoposide (40 × 10 –6 m , 0.5 h). C) IF staining was performed using anti‐ γ H2AX antibody. Representative images were shown (left panel). Scatter dot plot (right panel) represents the number of γ H2AX foci per cell. Data represent the mean ± SD of the number of γ H2AX foci from shNT‐expressing cells ( n = 80) and shPKM2‐expressing cells ( n = 81) treated with etoposide (Mann Whitney test). Data are representative of three independent experiments. D) IF staining was performed using anti‐MDC1 antibody. Representative images were shown (left panel). Scatter dot plot (right panel) represents the number of MDC1 foci per cell. Data represent mean ± SD of the MDC1 foci number of shNT‐expressing cells ( n = 75) or shPKM2‐expressing cells ( n = 81) treated with etoposide (Mann Whitney test). Data are representative of three independent experiments. E,F) U251 cells with or without PKM2 depletion were treated with or without etoposide (200 × 10 –6 m , 1 h). E) The chromatin fraction and F) nonchromatin‐bound fraction were prepared. G) U251 cells stably expressing shNT or shPKM2 were treated with or without etoposide (200 × 10 –6 m ) for indicated time. Whole‐cell lysates were prepared after sonication.

Article Snippet: Rabbit monoclonal antibodies against PKM2 (1:3000, 4053S), HA (1:3000, 3724S), β ‐actin (1:3000, 3700S), LaminB1 (1:3000, 12586s), H2AX pS139 (1:3000, 9718S), ATM pS1981 (1:1000, 13050S), ATM (1:1000, 2873), AKT pT308 (1:1000, 4056S), and rabbit polyclonal antibody against EGFR pY1068 (1:1000, 2234S) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Stable Transfection, Expressing, shRNA, Staining, Two Tailed Test, Single Cell Gel Electrophoresis, MANN-WHITNEY, Sonication

Journal: Advanced Science

Article Title: Pyruvate Facilitates FACT‐Mediated γ H2AX Loading to Chromatin and Promotes the Radiation Resistance of Glioblastoma

doi: 10.1002/advs.202104055

Figure Lengend Snippet: PKM2 interacts with FACT and enhances the interaction between FACT and nonchromatin bound γ H2AX. Immunoprecipitation (IP) and immunoblot (IB) analyses were performed with indicated antibodies. Data are representative of at least three independent experiments. A,B) U251 cells were transfected with A) Flag‐SPT16 or B) SFB‐SSRP1 and treated with etoposide (200 × 10 –6 m ) for indicated time. Whole‐cell lysates were prepared without sonication. C) U251 cells with or without SSRP1 depletion were treated with or without etoposide (200 × 10 –6 m , 1 h). The chromatin fraction was prepared. D,E) U251 cells stably expressing shNT or shPKM2 were infected with the lentivirus expressing D) SFB‐SSRP1 or E) SFB‐SPT16 and treated with or without etoposide (200 × 10 –6 m , 1 h). The nonchromatin‐bound fraction was prepared. F,G) PKM2‐depleted U251 cells were rescued with rPKM2 WT or K305Q and infected with the lentivirus expressing F) SFB‐SSRP1 or G) SFB‐SPT16. The cells were treated with or without etoposide (200 × 10 –6 m , 1 h) and the nonchromatin‐bound fraction was prepared. H) PKM2‐depleted U251 cells were rescued with rPKM2 WT or K305Q followed by etoposide treatment (200 × 10 –6 m , 1 h). Chromatin fraction (left panel) and nonchromatin‐bound fraction (right panel) were prepared.

Article Snippet: Rabbit monoclonal antibodies against PKM2 (1:3000, 4053S), HA (1:3000, 3724S), β ‐actin (1:3000, 3700S), LaminB1 (1:3000, 12586s), H2AX pS139 (1:3000, 9718S), ATM pS1981 (1:1000, 13050S), ATM (1:1000, 2873), AKT pT308 (1:1000, 4056S), and rabbit polyclonal antibody against EGFR pY1068 (1:1000, 2234S) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Immunoprecipitation, Western Blot, Transfection, Sonication, Stable Transfection, Expressing, Infection

Journal: Advanced Science

Article Title: Pyruvate Facilitates FACT‐Mediated γ H2AX Loading to Chromatin and Promotes the Radiation Resistance of Glioblastoma

doi: 10.1002/advs.202104055

Figure Lengend Snippet: Pyruvate increases the interaction between FACT and γ H2AX, γ H2AX levels in chromatin and tumor cell survival upon DNA damage. IP and IB analyses were performed with indicated antibodies. Data are representative of at least three independent experiments. A) U251 cells stably expressing shNT or shPKM2 were supplemented with or without pyruvate (10 × 10 –3 m , 3 h) and then treated with etoposide (200 × 10 –6 m , 1 h). Chromatin fraction (left panel) and nonchromatin‐bound fraction (right panel) were prepared. B) U251 cells stably expressing shNT or shPKM2 were supplemented with or without pyruvate (10 × 10 –3 m , 3 h) and then treated with etoposide (200 × 10 –6 m ) for indicated time. Cell viability was determined. Data represent the mean ± SD of the viability of the cells from three independent experiments (two‐tailed Student's t‐test). C) U251 cells stably expressing shNT or shPKM2 were supplemented with or without pyruvate (10 × 10 –3 m , 3 h) and then treated with or without etoposide (500 × 10 –6 m ) for 3 h. The representative images of the comet assay in these cells were shown (left panel). Scatter dot plot (right panel) represents the tail moments in the comet assay from cells treated with etoposide, expressing shNT with ( n = 75) or without ( n = 83) pyruvate, or cells expressing shPKM2 with ( n = 78) or without ( n = 78) pyruvate. Data represent mean ± SD of the tail moments (Mann Whitney test). Data are representative of three independent experiments. D,E) SSRP1 or SPT16 was immunoprecipitated and purified from U251 cells stably expressing D) SFB‐SSRP1 or E) Flag‐SPT16. γ H2AX was immunoprecipitated and purified from etoposide‐treated U251 cells. The in vitro pulldown experiment was performed by incubating the purified SSRP1 or SPT16 with purified γ H2AX in the absence or presence of pyruvate (1 × 10 –3 m ). PD: pulldown. F) U251 cells were supplemented with pyruvate (10 × 10 –3 m , 3 h) and then treated with etoposide (200 × 10 –6 m , 1 h). Chromatin fraction (left panel) and nonchromatin‐bound fraction (right panel) were prepared. G) U251 cells were supplemented with or without pyruvate (10 × 10 –3 m , 3 h) and then treated with etoposide (200 × 10 –6 m ) for indicated time. Cell viability was determined using Trypan blue staining. Data represent the mean ± SD of the viability of the cells from three independent experiments (two‐tailed Student's t‐test).

Article Snippet: Rabbit monoclonal antibodies against PKM2 (1:3000, 4053S), HA (1:3000, 3724S), β ‐actin (1:3000, 3700S), LaminB1 (1:3000, 12586s), H2AX pS139 (1:3000, 9718S), ATM pS1981 (1:1000, 13050S), ATM (1:1000, 2873), AKT pT308 (1:1000, 4056S), and rabbit polyclonal antibody against EGFR pY1068 (1:1000, 2234S) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Stable Transfection, Expressing, Two Tailed Test, Single Cell Gel Electrophoresis, MANN-WHITNEY, Immunoprecipitation, Purification, In Vitro, Staining

Journal: Advanced Science

Article Title: Pyruvate Facilitates FACT‐Mediated γ H2AX Loading to Chromatin and Promotes the Radiation Resistance of Glioblastoma

doi: 10.1002/advs.202104055

Figure Lengend Snippet: The binding of pyruvate to SSRP1 is required for FACT‐mediated γ H2AX loading to chromatin. IP and IB analyses were performed with indicated antibodies. Data are representative of at least three independent experiments. A) SPR (surface plasmon resonance) assay was performed with bacteria‐purified recombinant SSRP1 protein (100 µg mL –1 ) and increasing doses of pyruvate. K D : dissociation constant (pyruvate versus SSRP1). B) Metabolite–protein binding assay. Bacteria‐purified recombinant GST‐SSRP1 was incubated with 14 C‐labeled pyruvate. SSRP1‐bound pyruvate was quantified by scintillation counting. Data represent the mean ± SD of the relative metabolite amount bound to SSRP1 from three independent experiments (two‐tailed Student's t‐test). C) A representative image of the structure of human SSRP1 bound to pyruvate was shown. Atoms of pyruvate are presented as balls and sticks with carbon atoms in yellow and oxygen atoms in red. The whole protein is shown as a cartoon in green, while R54 is shown as sticks with purple carbon atoms and K73 is shown as lines with gray carbon atoms. Red dashed lines represent salt‐bridge and yellow ones for hydrogen bonds. The binding energy (Δ G bind ) of pyruvate with SSRP1 calculated using MM‐GBSA is ‐11 kcal mol –1 . D) Metabolite–protein binding assay. Bacteria‐purified recombinant GST‐SSRP1 WT or R54A was incubated with 14 C‐labeled pyruvate. The pyruvate bound to SSRP1 WT or R54A was quantified by scintillation counting. Data represent the mean ± SD of the relative metabolite amount bound to SSRP1 from three independent experiments (two‐tailed Student's t‐test). E) SSRP1 WT or R54A was immunoprecipitated and purified from U251 cells stably expressing SFB‐SSRP1 WT or R54A. γ H2AX was immunoprecipitated and purified from etoposide‐treated U251 cells. The in vitro pulldown experiment was performed by incubating the purified SSRP1 WT or R54A with purified γ H2AX in the absence or presence of pyruvate (1 × 10 –3 m ). F) HEK293T cells were transfected with SFB‐SSRP1 WT or R54A and then treated with or without etoposide (200 × 10 –6 m , 1 h). Nonchromatin‐bound fraction was prepared. G) In vitro histone exchange assay. H2AX‐H2B dimers were phosphorylated by purified recombinant monomeric ATM to obtain γ H2AX‐H2B dimers. Commercially purchased nucleosomes (120 ng) were incubated with γ H2AX‐H2B dimers, GST‐SPT16 and GST‐SSRP1 WT or R54A in the absence or presence of pyruvate (1 × 10 –3 m ) for 1 h. The recruitment of γ H2AX to the chromatin was determined by IB analysis of immobilized nucleosomes with anti‐ γ H2AX antibody. Histone H3 was used as loading control. H) U251 cells were infected with the lentivirus expressing shNT or shSSRP1 and the SSRP1‐depleted U251 cells were reconstituted with or without the expression of rSSRP1 WT or R54A. These cells were then treated with or without etoposide (200 × 10 –6 m , 1 h). Chromatin fraction (left panel) and non‐chromatin‐bound fraction (right panel) were prepared.

Article Snippet: Rabbit monoclonal antibodies against PKM2 (1:3000, 4053S), HA (1:3000, 3724S), β ‐actin (1:3000, 3700S), LaminB1 (1:3000, 12586s), H2AX pS139 (1:3000, 9718S), ATM pS1981 (1:1000, 13050S), ATM (1:1000, 2873), AKT pT308 (1:1000, 4056S), and rabbit polyclonal antibody against EGFR pY1068 (1:1000, 2234S) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Binding Assay, Chromatin Immunoprecipitation, SPR Assay, Bacteria, Purification, Recombinant, Protein Binding, Incubation, Labeling, Two Tailed Test, Immunoprecipitation, Stable Transfection, Expressing, In Vitro, Transfection, Control, Infection

Journal: Advanced Science

Article Title: Pyruvate Facilitates FACT‐Mediated γ H2AX Loading to Chromatin and Promotes the Radiation Resistance of Glioblastoma

doi: 10.1002/advs.202104055

Figure Lengend Snippet: PKM2 pS222 is required for DNA repair, tumor cell survival, and radiation resistance of glioblastoma. A–C) PKM2‐depleted U251 cells were rescued with rPKM2 WT or S222A. A) Cells were treated with etoposide (200 × 10 –6 m ) for indicated time. Cell viability was determined. Data represent the means ± SD of the viability of the cells from three independent experiments (two‐tailed Student's t‐test). B) The cells were treated with or without etoposide (500 × 10 –6 m , 3 h). The representative images of the comet assay in these cells were shown (left panel). Scatter dot plot (right panel) of the tail moments in the comet assay from shPKM2+rPKM2 WT cells ( n = 80) or shPKM2+rPKM2 S222A cells ( n = 80) treated with etoposide. Data represent mean ± SD of the tail moments (Mann Whitney test). Data are representative of three independent experiments. C) The cells were treated with etoposide (40 × 10 –6 m , 0.5 h). IF staining was performed using anti‐ γ H2AX antibody. Representative images were shown (left panel). Scatter dot plot (right panel) of the number of γ H2AX foci per cell. Data represent the mean ± SD of the number of γ H2AX foci from shPKM2+rPKM2 WT cells ( n = 76) and shPKM2+rPKM2 S222A cells ( n = 72) treated with etoposide (Mann Whitney test). Data are representative of three independent experiments. D–F) U87/EGFRvIII cells stably expressing luciferase were infected with the lentivirus expressing shNT or shPKM2 and the PKM2‐depleted U87/EGFRvIII cells were reconstituted with the expression of rPKM2 WT or S222A. These genetically modified cells (2 × 10 5 per mouse) were intracranially injected into randomized athymic nude mice (five mice per group) and then treated with or without IR (X‐ray) radiation (6 Gy). D) Bioluminescence imaging of tumor growth was carried out. E) Representative real‐time images were presented (left panel) and the intensities of luciferase were quantified (right panel) using living image software (PerkinElmer). F) Data represent the mean ± SD of luciferase intensity of five mice per group (two‐tailed Student's t‐test). Survival durations of these implanted mice were compared (Log‐rank test).

Article Snippet: Rabbit monoclonal antibodies against PKM2 (1:3000, 4053S), HA (1:3000, 3724S), β ‐actin (1:3000, 3700S), LaminB1 (1:3000, 12586s), H2AX pS139 (1:3000, 9718S), ATM pS1981 (1:1000, 13050S), ATM (1:1000, 2873), AKT pT308 (1:1000, 4056S), and rabbit polyclonal antibody against EGFR pY1068 (1:1000, 2234S) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Two Tailed Test, Single Cell Gel Electrophoresis, MANN-WHITNEY, Staining, Stable Transfection, Expressing, Luciferase, Infection, Genetically Modified, Injection, Imaging, Software

Journal: Advanced Science

Article Title: Pyruvate Facilitates FACT‐Mediated γ H2AX Loading to Chromatin and Promotes the Radiation Resistance of Glioblastoma

doi: 10.1002/advs.202104055

Figure Lengend Snippet: PKM2 pS222 levels correlate with the malignancy and prognosis of human glioblastoma. A) Survival of 76 patients with low (0–4.9 staining scores, blue curve) versus high (5.0–8 staining scores, red curve) PKM2 S222 phosphorylation levels (low, 25 patients; high, 51 patients) was compared (Log‐rank test). B) 73 diffuse astrocytoma (Grade II) specimens were immunohistochemically stained using anti‐PKM2 pS222 antibody. Staining scores of the specimens were compared with 48 stained GBM (Grade IV) specimens (two‐tailed Student's t‐test). C) Upon DNA damage, PKM2 is phosphorylated by AKT1 at serine (S) 222. Phosphorylated PKM2 interacts with the FACT complex and provides a local source of pyruvate. Pyruvate physically binds to SSRP1 and enhances the association of the FACT complex and γ H2AX, which facilitates FACT‐mediated chromatin loading of γ H2AX, thereby amplifying DNA damage signaling and promoting DNA damage repair.

Article Snippet: Rabbit monoclonal antibodies against PKM2 (1:3000, 4053S), HA (1:3000, 3724S), β ‐actin (1:3000, 3700S), LaminB1 (1:3000, 12586s), H2AX pS139 (1:3000, 9718S), ATM pS1981 (1:1000, 13050S), ATM (1:1000, 2873), AKT pT308 (1:1000, 4056S), and rabbit polyclonal antibody against EGFR pY1068 (1:1000, 2234S) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Staining, Phospho-proteomics, Two Tailed Test

Journal: Advanced Science

Article Title: Pyruvate Facilitates FACT‐Mediated γ H2AX Loading to Chromatin and Promotes the Radiation Resistance of Glioblastoma

doi: 10.1002/advs.202104055

Figure Lengend Snippet: PKM2 is required for γ H2AX levels in chromatin, DNA repair, and tumor cell survival. Immunoblotting (IB) experiments were performed with the indicated antibodies. Data are representative of at least three independent experiments. A) U251 cells stably expressing a nontargeting shRNA or PKM2 shRNA were rescued with or without rPKM2. Cells were treated with etoposide (200 × 10 –6 m ) for indicated time. Cell viability was determined using Trypan blue staining. Data represent the mean ± SD of the viability of the cells from three independent experiments (two‐tailed Student's t‐test). P values for comparisons between shNT and shPKM2 are shown in blue; comparisons between shPKM2 and shPKM2+rPKM2 are shown in green. B) U251 cells stably expressing shNT or shPKM2 were treated with or without etoposide (500 × 10 –6 m ) for 3 h. The representative images of the comet assay in these cells were shown (left panel). Scatter dot plot (right panel) of the tail moments in the comet assay from shNT‐expressing cells ( n = 86) or shPKM2‐expressing cells ( n = 86) treated with etoposide. Data represent mean ± SD of the tail moments (Mann Whitney test). Data are representative of three independent experiments. C,D) U251 cells stably expressing shNT or shPKM2 were treated with or without etoposide (40 × 10 –6 m , 0.5 h). C) IF staining was performed using anti‐ γ H2AX antibody. Representative images were shown (left panel). Scatter dot plot (right panel) represents the number of γ H2AX foci per cell. Data represent the mean ± SD of the number of γ H2AX foci from shNT‐expressing cells ( n = 80) and shPKM2‐expressing cells ( n = 81) treated with etoposide (Mann Whitney test). Data are representative of three independent experiments. D) IF staining was performed using anti‐MDC1 antibody. Representative images were shown (left panel). Scatter dot plot (right panel) represents the number of MDC1 foci per cell. Data represent mean ± SD of the MDC1 foci number of shNT‐expressing cells ( n = 75) or shPKM2‐expressing cells ( n = 81) treated with etoposide (Mann Whitney test). Data are representative of three independent experiments. E,F) U251 cells with or without PKM2 depletion were treated with or without etoposide (200 × 10 –6 m , 1 h). E) The chromatin fraction and F) nonchromatin‐bound fraction were prepared. G) U251 cells stably expressing shNT or shPKM2 were treated with or without etoposide (200 × 10 –6 m ) for indicated time. Whole‐cell lysates were prepared after sonication.

Article Snippet: Rabbit monoclonal antibodies against PKM2 (1:3000, 4053S), HA (1:3000, 3724S), β ‐actin (1:3000, 3700S), LaminB1 (1:3000, 12586s), H2AX pS139 (1:3000, 9718S), ATM pS1981 (1:1000, 13050S), ATM (1:1000, 2873), AKT pT308 (1:1000, 4056S), and rabbit polyclonal antibody against EGFR pY1068 (1:1000, 2234S) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Stable Transfection, Expressing, shRNA, Staining, Two Tailed Test, Single Cell Gel Electrophoresis, MANN-WHITNEY, Sonication

Journal: Advanced Science

Article Title: Pyruvate Facilitates FACT‐Mediated γ H2AX Loading to Chromatin and Promotes the Radiation Resistance of Glioblastoma

doi: 10.1002/advs.202104055

Figure Lengend Snippet: PKM2 interacts with FACT and enhances the interaction between FACT and nonchromatin bound γ H2AX. Immunoprecipitation (IP) and immunoblot (IB) analyses were performed with indicated antibodies. Data are representative of at least three independent experiments. A,B) U251 cells were transfected with A) Flag‐SPT16 or B) SFB‐SSRP1 and treated with etoposide (200 × 10 –6 m ) for indicated time. Whole‐cell lysates were prepared without sonication. C) U251 cells with or without SSRP1 depletion were treated with or without etoposide (200 × 10 –6 m , 1 h). The chromatin fraction was prepared. D,E) U251 cells stably expressing shNT or shPKM2 were infected with the lentivirus expressing D) SFB‐SSRP1 or E) SFB‐SPT16 and treated with or without etoposide (200 × 10 –6 m , 1 h). The nonchromatin‐bound fraction was prepared. F,G) PKM2‐depleted U251 cells were rescued with rPKM2 WT or K305Q and infected with the lentivirus expressing F) SFB‐SSRP1 or G) SFB‐SPT16. The cells were treated with or without etoposide (200 × 10 –6 m , 1 h) and the nonchromatin‐bound fraction was prepared. H) PKM2‐depleted U251 cells were rescued with rPKM2 WT or K305Q followed by etoposide treatment (200 × 10 –6 m , 1 h). Chromatin fraction (left panel) and nonchromatin‐bound fraction (right panel) were prepared.

Article Snippet: Rabbit monoclonal antibodies against PKM2 (1:3000, 4053S), HA (1:3000, 3724S), β ‐actin (1:3000, 3700S), LaminB1 (1:3000, 12586s), H2AX pS139 (1:3000, 9718S), ATM pS1981 (1:1000, 13050S), ATM (1:1000, 2873), AKT pT308 (1:1000, 4056S), and rabbit polyclonal antibody against EGFR pY1068 (1:1000, 2234S) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Immunoprecipitation, Western Blot, Transfection, Sonication, Stable Transfection, Expressing, Infection

Journal: Advanced Science

Article Title: Pyruvate Facilitates FACT‐Mediated γ H2AX Loading to Chromatin and Promotes the Radiation Resistance of Glioblastoma

doi: 10.1002/advs.202104055

Figure Lengend Snippet: AKT1‐dependent PKM2 S222 phosphorylation is necessary for PKM2 and FACT interaction upon DNA damage. IP and IB analyses were performed with indicated antibodies. Data are representative of at least three independent experiments. A,B) Co‐IP was performed with anti‐Flag antibody in HEK293T cells transfected with A) SFB‐SSRP1 or B) Flag‐SPT16 and HA‐PKM2 after etoposide treatment (200 × 10 –6 m , 1 h). The precipitated complex of SSRP1 or SPT16 was treated with or without CIP. C) HEK293T cells were transfected with Flag‐PKM2 WT or S222A and then treated with or without etoposide (200 × 10 –6 m , 1 h). D) U251, U87 cells, or normal human astrocyte cells (HA1800) stably expressing Flag‐PKM2 were treated with or without etoposide (200 × 10 –6 m , 1 h). E) HEK293T cells were transfected with SFB‐SSRP1 and HA‐PKM2 WT or S222A and then treated with or without etoposide (200 × 10 –6 m , 1 h). F) HEK293T cells were transfected with HA‐SPT16 and Flag‐PKM2 WT or S222A and then treated with or without etoposide (200 × 10 –6 m , 1 h). G,H) PKM2‐depleted HEK293T cells were rescued with rPKM2 WT or S222A. The cells were transfected with G) SFB‐SSRP1 or H) Flag‐SPT16 and then treated with or without etoposide (200 × 10 –6 m , 1 h). Co‐IP experiment was performed with anti‐Flag antibody in the nonchromatin‐bound protein fractions. I) PKM2‐depleted U251 cells were rescued with rPKM2 WT or S222A and then treated with or without etoposide (200 × 10 –6 m , 1 h). Chromatin fraction was prepared. J) U251 cells stably expressing Flag‐PKM2 were treated with or without ATM/DNA‐PK inhibitor Torin2 (100 × 10 –9 m ), EGFR inhibitor afatinib (5 × 10 –6 m ), PI3K inhibitor LY294002 (1 × 10 –6 m ), AKT1/2/3 inhibitor MK‐2206 (1 × 10 –6 m ), MEK1/2 inhibitor U0126 (5 × 10 –6 m ), JAK1/2 inhibitor ruxolitinib (50 × 10 –9 m ) or NF‐ κ B inhibitor BAY11‐7082 (50 × 10 –6 m ) for 12 h and then treated with or without etoposide (200 × 10 –6 m ) for 1 h. K) Co‐IP was performed with anti‐Flag antibody in HEK293T cells transfected with HA‐AKT1 and Flag‐PKM2 after etoposide treatment (200 × 10 –6 m , 1 h). L) In vitro kinase assays were carried out with purified recombinant His‐PKM2 and commercially purchased recombinant GST‐AKT1.

Article Snippet: Rabbit monoclonal antibodies against PKM2 (1:3000, 4053S), HA (1:3000, 3724S), β ‐actin (1:3000, 3700S), LaminB1 (1:3000, 12586s), H2AX pS139 (1:3000, 9718S), ATM pS1981 (1:1000, 13050S), ATM (1:1000, 2873), AKT pT308 (1:1000, 4056S), and rabbit polyclonal antibody against EGFR pY1068 (1:1000, 2234S) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Phospho-proteomics, Co-Immunoprecipitation Assay, Transfection, Stable Transfection, Expressing, In Vitro, Purification, Recombinant

Journal: Advanced Science

Article Title: Pyruvate Facilitates FACT‐Mediated γ H2AX Loading to Chromatin and Promotes the Radiation Resistance of Glioblastoma

doi: 10.1002/advs.202104055

Figure Lengend Snippet: PKM2 pS222 is required for DNA repair, tumor cell survival, and radiation resistance of glioblastoma. A–C) PKM2‐depleted U251 cells were rescued with rPKM2 WT or S222A. A) Cells were treated with etoposide (200 × 10 –6 m ) for indicated time. Cell viability was determined. Data represent the means ± SD of the viability of the cells from three independent experiments (two‐tailed Student's t‐test). B) The cells were treated with or without etoposide (500 × 10 –6 m , 3 h). The representative images of the comet assay in these cells were shown (left panel). Scatter dot plot (right panel) of the tail moments in the comet assay from shPKM2+rPKM2 WT cells ( n = 80) or shPKM2+rPKM2 S222A cells ( n = 80) treated with etoposide. Data represent mean ± SD of the tail moments (Mann Whitney test). Data are representative of three independent experiments. C) The cells were treated with etoposide (40 × 10 –6 m , 0.5 h). IF staining was performed using anti‐ γ H2AX antibody. Representative images were shown (left panel). Scatter dot plot (right panel) of the number of γ H2AX foci per cell. Data represent the mean ± SD of the number of γ H2AX foci from shPKM2+rPKM2 WT cells ( n = 76) and shPKM2+rPKM2 S222A cells ( n = 72) treated with etoposide (Mann Whitney test). Data are representative of three independent experiments. D–F) U87/EGFRvIII cells stably expressing luciferase were infected with the lentivirus expressing shNT or shPKM2 and the PKM2‐depleted U87/EGFRvIII cells were reconstituted with the expression of rPKM2 WT or S222A. These genetically modified cells (2 × 10 5 per mouse) were intracranially injected into randomized athymic nude mice (five mice per group) and then treated with or without IR (X‐ray) radiation (6 Gy). D) Bioluminescence imaging of tumor growth was carried out. E) Representative real‐time images were presented (left panel) and the intensities of luciferase were quantified (right panel) using living image software (PerkinElmer). F) Data represent the mean ± SD of luciferase intensity of five mice per group (two‐tailed Student's t‐test). Survival durations of these implanted mice were compared (Log‐rank test).

Article Snippet: Rabbit monoclonal antibodies against PKM2 (1:3000, 4053S), HA (1:3000, 3724S), β ‐actin (1:3000, 3700S), LaminB1 (1:3000, 12586s), H2AX pS139 (1:3000, 9718S), ATM pS1981 (1:1000, 13050S), ATM (1:1000, 2873), AKT pT308 (1:1000, 4056S), and rabbit polyclonal antibody against EGFR pY1068 (1:1000, 2234S) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Two Tailed Test, Single Cell Gel Electrophoresis, MANN-WHITNEY, Staining, Stable Transfection, Expressing, Luciferase, Infection, Genetically Modified, Injection, Imaging, Software

Journal: Advanced Science

Article Title: Pyruvate Facilitates FACT‐Mediated γ H2AX Loading to Chromatin and Promotes the Radiation Resistance of Glioblastoma

doi: 10.1002/advs.202104055

Figure Lengend Snippet: PKM2 pS222 levels correlate with the malignancy and prognosis of human glioblastoma. A) Survival of 76 patients with low (0–4.9 staining scores, blue curve) versus high (5.0–8 staining scores, red curve) PKM2 S222 phosphorylation levels (low, 25 patients; high, 51 patients) was compared (Log‐rank test). B) 73 diffuse astrocytoma (Grade II) specimens were immunohistochemically stained using anti‐PKM2 pS222 antibody. Staining scores of the specimens were compared with 48 stained GBM (Grade IV) specimens (two‐tailed Student's t‐test). C) Upon DNA damage, PKM2 is phosphorylated by AKT1 at serine (S) 222. Phosphorylated PKM2 interacts with the FACT complex and provides a local source of pyruvate. Pyruvate physically binds to SSRP1 and enhances the association of the FACT complex and γ H2AX, which facilitates FACT‐mediated chromatin loading of γ H2AX, thereby amplifying DNA damage signaling and promoting DNA damage repair.

Article Snippet: Rabbit monoclonal antibodies against PKM2 (1:3000, 4053S), HA (1:3000, 3724S), β ‐actin (1:3000, 3700S), LaminB1 (1:3000, 12586s), H2AX pS139 (1:3000, 9718S), ATM pS1981 (1:1000, 13050S), ATM (1:1000, 2873), AKT pT308 (1:1000, 4056S), and rabbit polyclonal antibody against EGFR pY1068 (1:1000, 2234S) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Staining, Phospho-proteomics, Two Tailed Test