Journal: Nature Communications

Article Title: Hypoxanthine phosphoribosyl transferase 1 metabolizes temozolomide to activate AMPK for driving chemoresistance of glioblastomas

doi: 10.1038/s41467-023-41663-2

Figure Lengend Snippet: a Cells were treated with 0.2 mM of 15 N-TMZ for 2 h. Intracellular 15 N-AICA were measured by HPLC-MS. Data represent the mean ± SD from sextuplicate experiments. ** P < 0.001. U87, Control vs. HPRT1 shRNA, P = 5.27e-10; WT vs. D138N, P = 1.63e-09; WT vs. K166A, P = 1.95e-09; MES28, Control vs. HPRT1 shRNA, P = 2.98e-11; WT vs. D138N, P = 2.61e-12; WT vs. K166A, P = 1.96e-12. b Cells were treated with 0.2 mM of 15 N-TMZ for 2 h. Intracellular 15 N-AICAR levels were measured by HPLC-MS. Data represent the mean ± SD from sextuplicate experiments. ** P < 0.001. U87, Control vs. HPRT1 shRNA, P = 4.75e-08; WT vs. D138N, P = 1.18e-10; WT vs. K166A, P = 1.32e-10; MES28, Control vs. HPRT1 shRNA, P = 1.61e-09; WT vs. D138N, P = 1.67e-08; WT vs. K166A, P = 1.31e-08. c Representative chromatograms of products of the HPRT1 kinase assay. d Michaelis-Menten curve of HPRT1 for AICA. Reactions were performed by mixing purified active HPRT1 and AICA. Data represent the mean ± SD from sextuplicate experiments. e and f Cells with or without shRNA-mediated HPRT1 depletion were treated with or without 0.2 mM of TMZ ( e ) or 0.25 of mM AICA for 2 h ( f ), respectively. Immunoblot analyses were performed with the indicated antibodies. Three biological repeats were repeated independently with similar results. g HPRT1-depleted cells with reconstituted expression of WT Flag-HPRT1, Flag-HPRT1 D138N, or Flag-HPRT1 K166A were treated with or without 0.2 mM of TMZ for 2 h. Immunoblot analyses were performed with the indicated antibodies. Three biological repeats were repeated independently with similar results. Statistics: a , b unpaired Student’s t -test for two-group comparison. Source data are provided as a Source Data file.

Article Snippet: Antibodies against ACC1 pS79 (11818), ACC1 (3676), AMPKα pT172 (50081), AMPKα (5831), AMPKα1 (4148), AMPK β1 (12063), γH2AX (9718), and α-Tubulin (3873) were purchased from Cell Signaling Technology (Beverly, MA, USA); Antibody against Flag (F3165) was purchased from Sigma-Aldrich (Shanghai, China); Antibodies against MGMT (ab108630) and UPRT (ab251653) were purchased from Abcam (Shanghai, China); Antibodies against APRT (21405-1-AP), OPRT (14830-1-AP), QPRT (25174-1-AP), HPRT1 (15059-1-AP), and RRM1 (10526-1-AP) were from Proteintech (Wuhan, China).

Techniques: Control, shRNA, Kinase Assay, Purification, Western Blot, Expressing, Comparison

Journal: Nature Communications

Article Title: Hypoxanthine phosphoribosyl transferase 1 metabolizes temozolomide to activate AMPK for driving chemoresistance of glioblastomas

doi: 10.1038/s41467-023-41663-2

Figure Lengend Snippet: a – e, g – i Immunoprecipitation and immunoblot analyses were performed with the indicated antibodies. a – i Three biological repeats were repeated independently with similar results. a Cells pretreated with or without 0.25 mM of AICA were treated with or without 0.2 mM of TMZ for the indicated time points. b Cells pretreated with or without 0.25 mM of A769662 were treated with or without 0.2 mM of TMZ for the indicated time points. c Cells were treated with or without 0.2 mM of TMZ for 2 h. d In vitro phosphorylation and SDS-PAGE analysis and autoradiography were performed by mixing purified WT His-RRM1 or His-RRM1 T52A protein with active AMPK in the presence of [γ- 32 P]ATP. e Bacterially purified WT His-RRM1 or His-RRM1 T52A was incubated with or without active AMPK in the presence or absence of ATP. f Stoichiometry of RRM1 phosphorylation by AMPK. Bacterially purified His-RRM1 was incubated with active AMPK in the presence of [γ- 32 P]ATP. The radioactive intensity of incorporated 32 P was measured and the incorporation of 32 P into RRM1 was calculated. Data represent the mean ± SD of triplicate samples. g The indicated cells expressing WT Flag-RRM1 or Flag-RRM1 T52A were treated with the indicated dose of TMZ for 2 h. h AMPKα1/2 double knockout (DKO) U87 cells expressing WT Flag-RRM1 or Flag-RRM1 T52A were treated with or without 0.2 mM of TMZ for 2 h. C1 and C2, two clones of AMPKα1/2 DKO U87 cells. i The indicated cells with or without HPRT1 depletion were transfected with vectors expressing WT Flag-RRM1 or Flag-RRM1 T52A. The cells were further treated with 0.2 mM of TMZ for 2 h.

Article Snippet: Antibodies against ACC1 pS79 (11818), ACC1 (3676), AMPKα pT172 (50081), AMPKα (5831), AMPKα1 (4148), AMPK β1 (12063), γH2AX (9718), and α-Tubulin (3873) were purchased from Cell Signaling Technology (Beverly, MA, USA); Antibody against Flag (F3165) was purchased from Sigma-Aldrich (Shanghai, China); Antibodies against MGMT (ab108630) and UPRT (ab251653) were purchased from Abcam (Shanghai, China); Antibodies against APRT (21405-1-AP), OPRT (14830-1-AP), QPRT (25174-1-AP), HPRT1 (15059-1-AP), and RRM1 (10526-1-AP) were from Proteintech (Wuhan, China).

Techniques: Immunoprecipitation, Western Blot, In Vitro, Phospho-proteomics, SDS Page, Autoradiography, Purification, Incubation, Expressing, Double Knockout, Clone Assay, Transfection

Journal: Nature Communications

Article Title: Hypoxanthine phosphoribosyl transferase 1 metabolizes temozolomide to activate AMPK for driving chemoresistance of glioblastomas

doi: 10.1038/s41467-023-41663-2

Figure Lengend Snippet: a and b WT His-RRM1 or His-RRM1 T52A mutant proteins were mixed with His-RRM2 and incubated with active AMPK in the presence of ATP for 20 min followed by HPLC-MS analysis. ( a ) Ribonucleotide reductase (RNR) activity was measured according to dCDP production. ( b ) K d was calculated. Data represent the mean ± SD from sextuplicate experiments. ** P < 0.001. a WT + AMPK vs. T52A + AMPK, P = 2.91e-08; b His-RRM1 WT vs. His-RRM1 WT + AMPK, P = 3.73e-05; His-RRM1 WT + AMPK vs. His-RRM1 T52A + AMPK, P = 0.00032. c Cells with or without HPRT1 depletion were transfected with Flag-RRM1 and treated with or without 0.2 mM of TMZ for 2 h. RNR activity was measured according to dCDP production. Immunoblotting analysis was performed to confirm the AMPK-mediated phosphorylation status of RRM1. Data represent the mean ± SD from sextuplicate experiments. ** P < 0.001. U87, Flag-RRM1 + TMZ vs. Flag-RRM1 + TMZ + HPRT1 shRNA, P = 3.18e-08; MES28, Flag-RRM1 + TMZ vs. Flag-RRM1 + TMZ + HPRT1 shRNA, P = 1.56e-11. d Cells with or without HPRT1 depletion were treated with or without 0.2 mM of TMZ, respectively, for 24 h. The rate of apoptotic cells was examined by FACS. Data represent the mean ± SD from sextuplicate experiments. ** P < 0.001. U87, TMZ vs. HPRT1 shRNA + TMZ, P = 1.12e-08; MES28, TMZ vs. HPRT1 shRNA + TMZ, P = 1.29e-06. e RRM1-depleted cells with reconstituted expression of Flag-RRM1 WT or Flag-RRM1 T52A were treated with or without 0.2 mM of TMZ, respectively, for 24 h. The rate of apoptotic cells was examined by FACS. Data represent the mean ± SD from sextuplicate experiments. ** P < 0.001. U87, Flag-RRM1 WT + TMZ vs. Flag-RRM1 T52A + TMZ, P = 3.68e-05; MES28, Flag-RRM1 WT + TMZ vs. Flag-RRM1 T52A + TMZ, P = 4.7e-09. Statistics: a – e unpaired Student’s t -test for two-group comparison. Source data are provided as a Source Data file.

Article Snippet: Antibodies against ACC1 pS79 (11818), ACC1 (3676), AMPKα pT172 (50081), AMPKα (5831), AMPKα1 (4148), AMPK β1 (12063), γH2AX (9718), and α-Tubulin (3873) were purchased from Cell Signaling Technology (Beverly, MA, USA); Antibody against Flag (F3165) was purchased from Sigma-Aldrich (Shanghai, China); Antibodies against MGMT (ab108630) and UPRT (ab251653) were purchased from Abcam (Shanghai, China); Antibodies against APRT (21405-1-AP), OPRT (14830-1-AP), QPRT (25174-1-AP), HPRT1 (15059-1-AP), and RRM1 (10526-1-AP) were from Proteintech (Wuhan, China).

Techniques: Mutagenesis, Incubation, Activity Assay, Transfection, Western Blot, Phospho-proteomics, shRNA, Expressing, Comparison

Journal: Nature Communications

Article Title: Hypoxanthine phosphoribosyl transferase 1 metabolizes temozolomide to activate AMPK for driving chemoresistance of glioblastomas

doi: 10.1038/s41467-023-41663-2

Figure Lengend Snippet: a Luciferase-expressing MES28 and U87 cells with or without HPRT1 depletion were intracranially injected into nude mice ( n = 10 for each group). Shown are luminescence intensity of tumors in representative mice at the indicated time points. b The survival time of the indicated groups of mice was recorded. ** P < 0.001. MES28, Control shRNA+TMZ vs. HPRT1 shRNA+TMZ, P = 1.97e-05; U87, Control shRNA+TMZ vs. HPRT1 shRNA+TMZ, P = 2.81e-05. c and d Representative IHC images of ACC1 pS79 ( c ) and γ-H2AX ( d ) were shown. Scale bars, 60 μm. n = 10 for each group. e Representative TUNEL images were shown. Scale bars, 60 μm. n = 10 for each group. f Luciferase-expressing MES28 and U87 cells with RRM1 depletion and reconstituted expression of WT Flag-RRM1 or the Flag-RRM1 T52A mutant were intracranially injected into nude mice ( n = 10 for each group). Shown are luminescence intensity of tumors in representative mice at the indicated time points. g The survival time of the indicated groups of mice was recorded. ** P < 0.001. MES28, Flag-RRM1 WT + TMZ vs. Flag-RRM1 T52A + TMZ, P = 4.42e-05; U87, Flag-RRM1 WT + TMZ vs. Flag-RRM1 T52A + TMZ, P = 0.00025. h Representative IHC images of RRM1 pT52 were shown. Scale bars, 60 μm. n = 10 for each group. i Representative TUNEL images were shown. Scale bars, 60 μm. n = 10 for each group. Statistics: b , g Log-rank test for two-group comparison. b Control shRNA + TMZ vs. HPRT1 shRNA + TMZ, g Flag-RRM1 WT + TMZ vs. Flag-RRM1 T52A + TMZ. Source data are provided as a Source Data file.

Article Snippet: Antibodies against ACC1 pS79 (11818), ACC1 (3676), AMPKα pT172 (50081), AMPKα (5831), AMPKα1 (4148), AMPK β1 (12063), γH2AX (9718), and α-Tubulin (3873) were purchased from Cell Signaling Technology (Beverly, MA, USA); Antibody against Flag (F3165) was purchased from Sigma-Aldrich (Shanghai, China); Antibodies against MGMT (ab108630) and UPRT (ab251653) were purchased from Abcam (Shanghai, China); Antibodies against APRT (21405-1-AP), OPRT (14830-1-AP), QPRT (25174-1-AP), HPRT1 (15059-1-AP), and RRM1 (10526-1-AP) were from Proteintech (Wuhan, China).

Techniques: Luciferase, Expressing, Injection, Control, shRNA, TUNEL Assay, Mutagenesis, Comparison

Journal: Nature Communications

Article Title: Hypoxanthine phosphoribosyl transferase 1 metabolizes temozolomide to activate AMPK for driving chemoresistance of glioblastomas

doi: 10.1038/s41467-023-41663-2

Figure Lengend Snippet: a and b Kaplan–Meier survival analysis based on HPRT1 expression from collected primary GBM samples ( a ) and the indicated GBM datasets ( b ). c Kaplan–Meier survival analysis based on HPRT1 expression from the indicated glioma datasets. d – f Kaplan–Meier survival analysis based on HPRT1 ( d ), AMPK pT172 ( e ), and RRM1 pT52 ( f ) expression from collected recurrent GBM samples. g – i The Pearson correlation test was used to analyze the correlation among HPRT1, AMPK pT172, and RRM1 pT52. Statistics: a – f Log-rank test for two-group comparison. Source data are provided as a Source Data file.

Article Snippet: Antibodies against ACC1 pS79 (11818), ACC1 (3676), AMPKα pT172 (50081), AMPKα (5831), AMPKα1 (4148), AMPK β1 (12063), γH2AX (9718), and α-Tubulin (3873) were purchased from Cell Signaling Technology (Beverly, MA, USA); Antibody against Flag (F3165) was purchased from Sigma-Aldrich (Shanghai, China); Antibodies against MGMT (ab108630) and UPRT (ab251653) were purchased from Abcam (Shanghai, China); Antibodies against APRT (21405-1-AP), OPRT (14830-1-AP), QPRT (25174-1-AP), HPRT1 (15059-1-AP), and RRM1 (10526-1-AP) were from Proteintech (Wuhan, China).

Techniques: Expressing, Comparison