soluble cd59  (Hycult Biotech)

Journal: bioRxiv

Article Title: Activation of the mevalonate pathway in response to anti-cancer treatments drives glioblastoma recurrences through activation of Rac-1

doi: 10.1101/2023.07.23.550205

Figure Lengend Snippet: (a/b) Western blotting of p -ERK, total ERK, p -P38, and total P38 in patient-derived GBM HK374 and HK217 cell lines upon radiation (a single dose of 10 Gy) in combination of QTP (10 µM) or TMZ (1 mM) or Vincristine (250 nM) at 2 hours after treatment. (c) The densitometry measurements of p -ERK/total ERK and p -P38/total P38 using Image J. (d/e) Heatmap showing the results of quantitative RT-PCR for the cholesterol biosynthesis related genes in both HK374 and HK217 cells treated with radiation (a single dose of 4 Gy) in the presence of absence of TMZ (1 mM) for two consecutive days. ( f/g ) Heatmap showing the results of quantitative RT-PCR for the cholesterol biosynthesis related genes in both HK374 and HK217 cells treated with radiation (a single dose of 4 Gy) in the presence of absence of Vincristine (250 nM) for 24 hours. All experiments have been performed with at least 3 biological independent repeats. p- values were calculated using One-way ANOVA. * p -value < 0.05, ** p -value < 0.01, *** p -value < 0.001, **** p -value < 0.0001.

Article Snippet: HK374 cells were seeded into 6-well tissue-culture plates and transfected with scramble siRNA or Rac1 siRNA by incubation with RNAiMAX-siRNA Lipofectamine duplex (#13778075, Thermo Fisher Scientific) in Opti-MEM medium overnight at 37°C with 5% CO 2.

Techniques: Western Blot, Derivative Assay, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Activation of the mevalonate pathway in response to anti-cancer treatments drives glioblastoma recurrences through activation of Rac-1

doi: 10.1101/2023.07.23.550205

Figure Lengend Snippet: (a/b) Brain and plasma levels of atorvastatin and simvastatin in C57BL/6 mice after a single injection (atorvastatin – 30 mg/kg, i.p.; simvastatin – 7 mg/kg, i.p.). (c) The levels of total, free cholesterol and cholesterol-esters in the tumors harvested from the PDOX GBM mouse model at day 5 after the treatment of radiation (a single dose of 4 Gy) in combination with QTP (30 mg/kg), atorvastatin (30 mg/kg) or simvastatin (7 mg/kg). (d) The concentration of free fatty acid in the tumors harvested from the PDOX GBM mouse model at day 5 after the treatments. (e) Heatmap showing the results of quantitative RT-PCR for the cholesterol biosynthesis related genes in the tumor specimen harvested from the PDOX GBM mouse model at day 5 after the treatments using the human- and mouse-specific primers. (f) Survival curves for NSG mice implanted intracranially with 3x10 5 HK374-GFP-Luciferase glioma cells and grafted for 3 days. Mice were irradiated with a single fraction of 0 or 10 Gy and treated with corn oil or QTP (30 mg/kg, subQ, 5-day on/2-day off schedule) or triple combination of 10 Gy plus QTP and simvastatin (7 mg/kg, i.p., 5-day on/2-day off schedule) continuously until they reached the study endpoint. Log-rank (Mantel-Cox) test for comparison of Kaplan-Meier survival curves. (g) Weight curves for the NSG mice in different treatment groups. All experiments have been performed with at least 3 biological independent repeats. p- values were calculated using One-way ANOVA. * p -value < 0.05, **** p -value < 0.0001, ns: not significant.

Article Snippet: HK374 cells were seeded into 6-well tissue-culture plates and transfected with scramble siRNA or Rac1 siRNA by incubation with RNAiMAX-siRNA Lipofectamine duplex (#13778075, Thermo Fisher Scientific) in Opti-MEM medium overnight at 37°C with 5% CO 2.

Techniques: Injection, Concentration Assay, Quantitative RT-PCR, Luciferase, Irradiation, Comparison

Journal: bioRxiv

Article Title: Activation of the mevalonate pathway in response to anti-cancer treatments drives glioblastoma recurrences through activation of Rac-1

doi: 10.1101/2023.07.23.550205

Figure Lengend Snippet: (a) Heat map showing the results of quantitative RT-PCR for the cholesterol biosynthesis related genes in HK374 cells treated with radiation (a single dose of 4 Gy) in the presence of absence of ONC201 (a single treatment of 2.5 µM) at 48 and 72 hours. (b) Survival curves for C57BL/6 mice implanted intracranially with 2x10 5 GL261-GFP-Luciferase mouse glioma cells and grafted for 7 days. Mice were irradiated with a single fraction of 0 or 10 Gy and weekly treated with Saline or ONC201 (50 mg/kg, i.p.) or triple combination of 10 Gy plus ONC201 (weekly) and atorvastatin (30 mg/kg, i.p., 5-day on/2-day off schedule) continuously until they reached the study endpoint. Log-rank (Mantel-Cox) test for comparison of Kaplan-Meier survival curves. (c) H&E-stained coronal sections of the C57BL/6 mice brains implanted with GL261-GFP-Luc cells which were irradiated at 10 Gy and treated continuously with ONC201 in the presence of absence of atorvastatin until they met the criteria for study endpoint. (d) Schematic of the experimental design of fractionated irradiation in syngeneic mouse model of GBM. (e) Kaplan-Meier survival curves for C57BL/6 mice implanted intracranially with GL261-GFP-Luciferase mouse glioma cells and treated with either a single fraction of 0 or 10 Gy or 5 daily fractions of 3 Gy each and daily doses of either corn oil, QTP (30 mg/kg, subQ), or QTP plus atorvastatin (30 mg/kg, i.p.). After completion of the radiation treatment all animals were treated with QTP plus atorvastatin until they reached criteria for euthanasia. Log-rank (Mantel-Cox) test for comparison of Kaplan-Meier survival curves. (f) H&E stained coronal sections of the C57BL/6 mice brains from the groups of 5 x 3 Gy -> QTP + atorvastatin, 5 x 3 Gy + QTP -> QTP + atorvastatin and 5 x 3 Gy + QTP + atorvastatin -> QTP + atorvastatin.

Article Snippet: HK374 cells were seeded into 6-well tissue-culture plates and transfected with scramble siRNA or Rac1 siRNA by incubation with RNAiMAX-siRNA Lipofectamine duplex (#13778075, Thermo Fisher Scientific) in Opti-MEM medium overnight at 37°C with 5% CO 2.

Techniques: Quantitative RT-PCR, Luciferase, Irradiation, Saline, Comparison, Staining

Journal: bioRxiv

Article Title: Activation of the mevalonate pathway in response to anti-cancer treatments drives glioblastoma recurrences through activation of Rac-1

doi: 10.1101/2023.07.23.550205

Figure Lengend Snippet: ( a ) Cholesterol biosynthesis pathway and the key selected inhibitors. ( b-e ) Sphere-forming capacity of HK374 spheres treated either with GGTI-298 (GGTase inhibitor) or YM-53601 (squalene synthase inhibitor) or Zaragozic acid (squalene synthase inhibitor) or Lonafarnib (farnesyltransferase inhibitor) at 100, 500 nM, 1 µM concentrations when combined with radiation (a single dose of 4 Gy) and QTP (10 µM). ( f-i ) Sphere-forming capacity of HK374 spheres treated either with Ehop-016 (Rac GTPase inhibitor) or Rhosin (RhoA-specific inhibitor) or Y27632 (ROCK1/2 inhibitor) or CID44216842 (Cdc42-selective inhibitor) at 500 nM, 1, 5, 10 µM concentrations when combined with radiation and QTP. All experiments have been performed with at least 3 biological independent repeats. p- values were calculated using One-way ANOVA. * p -value < 0.05, ** p -value < 0.01, *** p -value < 0.001, **** p -value < 0.0001.

Article Snippet: HK374 cells were seeded into 6-well tissue-culture plates and transfected with scramble siRNA or Rac1 siRNA by incubation with RNAiMAX-siRNA Lipofectamine duplex (#13778075, Thermo Fisher Scientific) in Opti-MEM medium overnight at 37°C with 5% CO 2.

Techniques:

Journal: bioRxiv

Article Title: Activation of the mevalonate pathway in response to anti-cancer treatments drives glioblastoma recurrences through activation of Rac-1

doi: 10.1101/2023.07.23.550205

Figure Lengend Snippet: (a) HK374 cells were treated with 0 or 4 Gy in the presence or absence of QTP (10 µM) and/or atorvastatin (1 µM) for 48 hours. The activated Rac1 was immunoprecipitated by 10 µg PAK-PBD agarose beads from the whole cell lysates and subjected to immunoblotting against Rac1, along with the total proteins. His-tagged Rac1 protein serves as the positive control. (b) The densitometry measurements of activated Rac1/total Rac1 using Image J. (c) Transwell migration assay of HK374 cells pre-treated with 0 or 4 Gy in the presence or absence of QTP (10 µM) and/or atorvastatin (1 µM) for 48 hours. (d) The quantification of migrated cells using Image J. (e) Confocal images of microtubules in HK374 cells treated with 0 or 4 Gy in the presence or absence of QTP (10 µM) and/or atorvastatin (1 µM) for 48 hours. White arrowheads: filopodia. Yellow arrowheads: tunneling nanotubes (TNTs). (f/g) Rac1 knock-down efficiency was evaluated at both mRNA (qRT-PCR) and protein (western blotting) levels at day 2 and day 7 after siRNA transfection. β-actin was used as the loading control and the densitometry measurements of Rac1 using Image J. (h) Clonogenic assay of siCtrl or siRac1 transfected HK374 cells treated with 0 or 4 Gy in the presence or absence of QTP (10 µM) and/or atorvastatin (1 µM) for 7 days. (i) Sphere-forming capacity of siCtrl or siRac1 HK374 spheres treated with 0 or 4 Gy in the presence or absence of QTP (10 µM) and/or atorvastatin (1 µM). All experiments have been performed with at least 3 biological independent repeats. p- values were calculated using One-way ANOVA for b, d ; Unpaired Student’s t-tests for f, g ; Two-way ANOVA for i . * p -value < 0.05, ** p -value < 0.01, *** p -value < 0.001, **** p -value < 0.0001, ns: not significant.

Article Snippet: HK374 cells were seeded into 6-well tissue-culture plates and transfected with scramble siRNA or Rac1 siRNA by incubation with RNAiMAX-siRNA Lipofectamine duplex (#13778075, Thermo Fisher Scientific) in Opti-MEM medium overnight at 37°C with 5% CO 2.

Techniques: Immunoprecipitation, Western Blot, Positive Control, Transwell Migration Assay, Knockdown, Quantitative RT-PCR, Transfection, Control, Clonogenic Assay

Journal: bioRxiv

Article Title: Activation of the mevalonate pathway in response to anti-cancer treatments drives glioblastoma recurrences through activation of Rac-1

doi: 10.1101/2023.07.23.550205

Figure Lengend Snippet: (a/b) Western blotting of p -ERK, total ERK, p -P38, and total P38 in patient-derived GBM HK374 and HK217 cell lines upon radiation (a single dose of 10 Gy) in combination of QTP (10 µM) or TMZ (1 mM) or Vincristine (250 nM) at 2 hours after treatment. (c) The densitometry measurements of p -ERK/total ERK and p -P38/total P38 using Image J. (d/e) Heatmap showing the results of quantitative RT-PCR for the cholesterol biosynthesis related genes in both HK374 and HK217 cells treated with radiation (a single dose of 4 Gy) in the presence of absence of TMZ (1 mM) for two consecutive days. ( f/g ) Heatmap showing the results of quantitative RT-PCR for the cholesterol biosynthesis related genes in both HK374 and HK217 cells treated with radiation (a single dose of 4 Gy) in the presence of absence of Vincristine (250 nM) for 24 hours. All experiments have been performed with at least 3 biological independent repeats. p- values were calculated using One-way ANOVA. * p -value < 0.05, ** p -value < 0.01, *** p -value < 0.001, **** p -value < 0.0001.

Article Snippet: HK374 cells were plated into 35 mm dish (No. 1.5 Coverslip, 10 mm Glass Diameter, Poly-D-Lysine coated; #P35GC-1.5-10-C, MatTek Ashland, MA) and the following day treated with QTP (10 µM) or QTP + atorvastatin (1 µM) and irradiated with a single dose of 4 Gy one hour after the drug treatment.

Techniques: Western Blot, Derivative Assay, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Activation of the mevalonate pathway in response to anti-cancer treatments drives glioblastoma recurrences through activation of Rac-1

doi: 10.1101/2023.07.23.550205

Figure Lengend Snippet: (a/b) Brain and plasma levels of atorvastatin and simvastatin in C57BL/6 mice after a single injection (atorvastatin – 30 mg/kg, i.p.; simvastatin – 7 mg/kg, i.p.). (c) The levels of total, free cholesterol and cholesterol-esters in the tumors harvested from the PDOX GBM mouse model at day 5 after the treatment of radiation (a single dose of 4 Gy) in combination with QTP (30 mg/kg), atorvastatin (30 mg/kg) or simvastatin (7 mg/kg). (d) The concentration of free fatty acid in the tumors harvested from the PDOX GBM mouse model at day 5 after the treatments. (e) Heatmap showing the results of quantitative RT-PCR for the cholesterol biosynthesis related genes in the tumor specimen harvested from the PDOX GBM mouse model at day 5 after the treatments using the human- and mouse-specific primers. (f) Survival curves for NSG mice implanted intracranially with 3x10 5 HK374-GFP-Luciferase glioma cells and grafted for 3 days. Mice were irradiated with a single fraction of 0 or 10 Gy and treated with corn oil or QTP (30 mg/kg, subQ, 5-day on/2-day off schedule) or triple combination of 10 Gy plus QTP and simvastatin (7 mg/kg, i.p., 5-day on/2-day off schedule) continuously until they reached the study endpoint. Log-rank (Mantel-Cox) test for comparison of Kaplan-Meier survival curves. (g) Weight curves for the NSG mice in different treatment groups. All experiments have been performed with at least 3 biological independent repeats. p- values were calculated using One-way ANOVA. * p -value < 0.05, **** p -value < 0.0001, ns: not significant.

Article Snippet: HK374 cells were plated into 35 mm dish (No. 1.5 Coverslip, 10 mm Glass Diameter, Poly-D-Lysine coated; #P35GC-1.5-10-C, MatTek Ashland, MA) and the following day treated with QTP (10 µM) or QTP + atorvastatin (1 µM) and irradiated with a single dose of 4 Gy one hour after the drug treatment.

Techniques: Injection, Concentration Assay, Quantitative RT-PCR, Luciferase, Irradiation

Journal: bioRxiv

Article Title: Activation of the mevalonate pathway in response to anti-cancer treatments drives glioblastoma recurrences through activation of Rac-1

doi: 10.1101/2023.07.23.550205

Figure Lengend Snippet: (a) Heat map showing the results of quantitative RT-PCR for the cholesterol biosynthesis related genes in HK374 cells treated with radiation (a single dose of 4 Gy) in the presence of absence of ONC201 (a single treatment of 2.5 µM) at 48 and 72 hours. (b) Survival curves for C57BL/6 mice implanted intracranially with 2x10 5 GL261-GFP-Luciferase mouse glioma cells and grafted for 7 days. Mice were irradiated with a single fraction of 0 or 10 Gy and weekly treated with Saline or ONC201 (50 mg/kg, i.p.) or triple combination of 10 Gy plus ONC201 (weekly) and atorvastatin (30 mg/kg, i.p., 5-day on/2-day off schedule) continuously until they reached the study endpoint. Log-rank (Mantel-Cox) test for comparison of Kaplan-Meier survival curves. (c) H&E-stained coronal sections of the C57BL/6 mice brains implanted with GL261-GFP-Luc cells which were irradiated at 10 Gy and treated continuously with ONC201 in the presence of absence of atorvastatin until they met the criteria for study endpoint. (d) Schematic of the experimental design of fractionated irradiation in syngeneic mouse model of GBM. (e) Kaplan-Meier survival curves for C57BL/6 mice implanted intracranially with GL261-GFP-Luciferase mouse glioma cells and treated with either a single fraction of 0 or 10 Gy or 5 daily fractions of 3 Gy each and daily doses of either corn oil, QTP (30 mg/kg, subQ), or QTP plus atorvastatin (30 mg/kg, i.p.). After completion of the radiation treatment all animals were treated with QTP plus atorvastatin until they reached criteria for euthanasia. Log-rank (Mantel-Cox) test for comparison of Kaplan-Meier survival curves. (f) H&E stained coronal sections of the C57BL/6 mice brains from the groups of 5 x 3 Gy -> QTP + atorvastatin, 5 x 3 Gy + QTP -> QTP + atorvastatin and 5 x 3 Gy + QTP + atorvastatin -> QTP + atorvastatin.

Article Snippet: HK374 cells were plated into 35 mm dish (No. 1.5 Coverslip, 10 mm Glass Diameter, Poly-D-Lysine coated; #P35GC-1.5-10-C, MatTek Ashland, MA) and the following day treated with QTP (10 µM) or QTP + atorvastatin (1 µM) and irradiated with a single dose of 4 Gy one hour after the drug treatment.

Techniques: Quantitative RT-PCR, Luciferase, Irradiation, Staining

Journal: bioRxiv

Article Title: Activation of the mevalonate pathway in response to anti-cancer treatments drives glioblastoma recurrences through activation of Rac-1

doi: 10.1101/2023.07.23.550205

Figure Lengend Snippet: ( a ) Cholesterol biosynthesis pathway and the key selected inhibitors. ( b-e ) Sphere-forming capacity of HK374 spheres treated either with GGTI-298 (GGTase inhibitor) or YM-53601 (squalene synthase inhibitor) or Zaragozic acid (squalene synthase inhibitor) or Lonafarnib (farnesyltransferase inhibitor) at 100, 500 nM, 1 µM concentrations when combined with radiation (a single dose of 4 Gy) and QTP (10 µM). ( f-i ) Sphere-forming capacity of HK374 spheres treated either with Ehop-016 (Rac GTPase inhibitor) or Rhosin (RhoA-specific inhibitor) or Y27632 (ROCK1/2 inhibitor) or CID44216842 (Cdc42-selective inhibitor) at 500 nM, 1, 5, 10 µM concentrations when combined with radiation and QTP. All experiments have been performed with at least 3 biological independent repeats. p- values were calculated using One-way ANOVA. * p -value < 0.05, ** p -value < 0.01, *** p -value < 0.001, **** p -value < 0.0001.

Article Snippet: HK374 cells were plated into 35 mm dish (No. 1.5 Coverslip, 10 mm Glass Diameter, Poly-D-Lysine coated; #P35GC-1.5-10-C, MatTek Ashland, MA) and the following day treated with QTP (10 µM) or QTP + atorvastatin (1 µM) and irradiated with a single dose of 4 Gy one hour after the drug treatment.

Techniques:

Journal: bioRxiv

Article Title: Activation of the mevalonate pathway in response to anti-cancer treatments drives glioblastoma recurrences through activation of Rac-1

doi: 10.1101/2023.07.23.550205

Figure Lengend Snippet: (a) HK374 cells were treated with 0 or 4 Gy in the presence or absence of QTP (10 µM) and/or atorvastatin (1 µM) for 48 hours. The activated Rac1 was immunoprecipitated by 10 µg PAK-PBD agarose beads from the whole cell lysates and subjected to immunoblotting against Rac1, along with the total proteins. His-tagged Rac1 protein serves as the positive control. (b) The densitometry measurements of activated Rac1/total Rac1 using Image J. (c) Transwell migration assay of HK374 cells pre-treated with 0 or 4 Gy in the presence or absence of QTP (10 µM) and/or atorvastatin (1 µM) for 48 hours. (d) The quantification of migrated cells using Image J. (e) Confocal images of microtubules in HK374 cells treated with 0 or 4 Gy in the presence or absence of QTP (10 µM) and/or atorvastatin (1 µM) for 48 hours. White arrowheads: filopodia. Yellow arrowheads: tunneling nanotubes (TNTs). (f/g) Rac1 knock-down efficiency was evaluated at both mRNA (qRT-PCR) and protein (western blotting) levels at day 2 and day 7 after siRNA transfection. β-actin was used as the loading control and the densitometry measurements of Rac1 using Image J. (h) Clonogenic assay of siCtrl or siRac1 transfected HK374 cells treated with 0 or 4 Gy in the presence or absence of QTP (10 µM) and/or atorvastatin (1 µM) for 7 days. (i) Sphere-forming capacity of siCtrl or siRac1 HK374 spheres treated with 0 or 4 Gy in the presence or absence of QTP (10 µM) and/or atorvastatin (1 µM). All experiments have been performed with at least 3 biological independent repeats. p- values were calculated using One-way ANOVA for b, d ; Unpaired Student’s t-tests for f, g ; Two-way ANOVA for i . * p -value < 0.05, ** p -value < 0.01, *** p -value < 0.001, **** p -value < 0.0001, ns: not significant.

Article Snippet: HK374 cells were plated into 35 mm dish (No. 1.5 Coverslip, 10 mm Glass Diameter, Poly-D-Lysine coated; #P35GC-1.5-10-C, MatTek Ashland, MA) and the following day treated with QTP (10 µM) or QTP + atorvastatin (1 µM) and irradiated with a single dose of 4 Gy one hour after the drug treatment.

Techniques: Immunoprecipitation, Western Blot, Positive Control, Transwell Migration Assay, Quantitative RT-PCR, Transfection, Clonogenic Assay

Journal: bioRxiv

Article Title: Activation of the mevalonate pathway in response to anti-cancer treatments drives glioblastoma recurrences through activation of Rac-1

doi: 10.1101/2023.07.23.550205

Figure Lengend Snippet: (a/b) Western blotting of p -ERK, total ERK, p -P38, and total P38 in patient-derived GBM HK374 and HK217 cell lines upon radiation (a single dose of 10 Gy) in combination of QTP (10 µM) or TMZ (1 mM) or Vincristine (250 nM) at 2 hours after treatment. (c) The densitometry measurements of p -ERK/total ERK and p -P38/total P38 using Image J. (d/e) Heatmap showing the results of quantitative RT-PCR for the cholesterol biosynthesis related genes in both HK374 and HK217 cells treated with radiation (a single dose of 4 Gy) in the presence of absence of TMZ (1 mM) for two consecutive days. ( f/g ) Heatmap showing the results of quantitative RT-PCR for the cholesterol biosynthesis related genes in both HK374 and HK217 cells treated with radiation (a single dose of 4 Gy) in the presence of absence of Vincristine (250 nM) for 24 hours. All experiments have been performed with at least 3 biological independent repeats. p- values were calculated using One-way ANOVA. * p -value < 0.05, ** p -value < 0.01, *** p -value < 0.001, **** p -value < 0.0001.

Article Snippet: To explore the activation of MAPK pathway in vitro , HK374 cells were serum starved and the following day treated with a single dose of 10 Gy in the absence or presence of QTP (10 µM), temozolomide (1 mM; #14163, Cayman Chemical) or Vincristine (250 nM; #HY-N0488, MedChem Express, Monmouth Junction, NJ).

Techniques: Western Blot, Derivative Assay, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Activation of the mevalonate pathway in response to anti-cancer treatments drives glioblastoma recurrences through activation of Rac-1

doi: 10.1101/2023.07.23.550205

Figure Lengend Snippet: (a/b) Brain and plasma levels of atorvastatin and simvastatin in C57BL/6 mice after a single injection (atorvastatin – 30 mg/kg, i.p.; simvastatin – 7 mg/kg, i.p.). (c) The levels of total, free cholesterol and cholesterol-esters in the tumors harvested from the PDOX GBM mouse model at day 5 after the treatment of radiation (a single dose of 4 Gy) in combination with QTP (30 mg/kg), atorvastatin (30 mg/kg) or simvastatin (7 mg/kg). (d) The concentration of free fatty acid in the tumors harvested from the PDOX GBM mouse model at day 5 after the treatments. (e) Heatmap showing the results of quantitative RT-PCR for the cholesterol biosynthesis related genes in the tumor specimen harvested from the PDOX GBM mouse model at day 5 after the treatments using the human- and mouse-specific primers. (f) Survival curves for NSG mice implanted intracranially with 3x10 5 HK374-GFP-Luciferase glioma cells and grafted for 3 days. Mice were irradiated with a single fraction of 0 or 10 Gy and treated with corn oil or QTP (30 mg/kg, subQ, 5-day on/2-day off schedule) or triple combination of 10 Gy plus QTP and simvastatin (7 mg/kg, i.p., 5-day on/2-day off schedule) continuously until they reached the study endpoint. Log-rank (Mantel-Cox) test for comparison of Kaplan-Meier survival curves. (g) Weight curves for the NSG mice in different treatment groups. All experiments have been performed with at least 3 biological independent repeats. p- values were calculated using One-way ANOVA. * p -value < 0.05, **** p -value < 0.0001, ns: not significant.

Article Snippet: To explore the activation of MAPK pathway in vitro , HK374 cells were serum starved and the following day treated with a single dose of 10 Gy in the absence or presence of QTP (10 µM), temozolomide (1 mM; #14163, Cayman Chemical) or Vincristine (250 nM; #HY-N0488, MedChem Express, Monmouth Junction, NJ).

Techniques: Injection, Concentration Assay, Quantitative RT-PCR, Luciferase, Irradiation, Comparison

Journal: bioRxiv

Article Title: Activation of the mevalonate pathway in response to anti-cancer treatments drives glioblastoma recurrences through activation of Rac-1

doi: 10.1101/2023.07.23.550205

Figure Lengend Snippet: (a) Heat map showing the results of quantitative RT-PCR for the cholesterol biosynthesis related genes in HK374 cells treated with radiation (a single dose of 4 Gy) in the presence of absence of ONC201 (a single treatment of 2.5 µM) at 48 and 72 hours. (b) Survival curves for C57BL/6 mice implanted intracranially with 2x10 5 GL261-GFP-Luciferase mouse glioma cells and grafted for 7 days. Mice were irradiated with a single fraction of 0 or 10 Gy and weekly treated with Saline or ONC201 (50 mg/kg, i.p.) or triple combination of 10 Gy plus ONC201 (weekly) and atorvastatin (30 mg/kg, i.p., 5-day on/2-day off schedule) continuously until they reached the study endpoint. Log-rank (Mantel-Cox) test for comparison of Kaplan-Meier survival curves. (c) H&E-stained coronal sections of the C57BL/6 mice brains implanted with GL261-GFP-Luc cells which were irradiated at 10 Gy and treated continuously with ONC201 in the presence of absence of atorvastatin until they met the criteria for study endpoint. (d) Schematic of the experimental design of fractionated irradiation in syngeneic mouse model of GBM. (e) Kaplan-Meier survival curves for C57BL/6 mice implanted intracranially with GL261-GFP-Luciferase mouse glioma cells and treated with either a single fraction of 0 or 10 Gy or 5 daily fractions of 3 Gy each and daily doses of either corn oil, QTP (30 mg/kg, subQ), or QTP plus atorvastatin (30 mg/kg, i.p.). After completion of the radiation treatment all animals were treated with QTP plus atorvastatin until they reached criteria for euthanasia. Log-rank (Mantel-Cox) test for comparison of Kaplan-Meier survival curves. (f) H&E stained coronal sections of the C57BL/6 mice brains from the groups of 5 x 3 Gy -> QTP + atorvastatin, 5 x 3 Gy + QTP -> QTP + atorvastatin and 5 x 3 Gy + QTP + atorvastatin -> QTP + atorvastatin.

Article Snippet: To explore the activation of MAPK pathway in vitro , HK374 cells were serum starved and the following day treated with a single dose of 10 Gy in the absence or presence of QTP (10 µM), temozolomide (1 mM; #14163, Cayman Chemical) or Vincristine (250 nM; #HY-N0488, MedChem Express, Monmouth Junction, NJ).

Techniques: Quantitative RT-PCR, Luciferase, Irradiation, Saline, Comparison, Staining

Journal: bioRxiv

Article Title: Activation of the mevalonate pathway in response to anti-cancer treatments drives glioblastoma recurrences through activation of Rac-1

doi: 10.1101/2023.07.23.550205

Figure Lengend Snippet: ( a ) Cholesterol biosynthesis pathway and the key selected inhibitors. ( b-e ) Sphere-forming capacity of HK374 spheres treated either with GGTI-298 (GGTase inhibitor) or YM-53601 (squalene synthase inhibitor) or Zaragozic acid (squalene synthase inhibitor) or Lonafarnib (farnesyltransferase inhibitor) at 100, 500 nM, 1 µM concentrations when combined with radiation (a single dose of 4 Gy) and QTP (10 µM). ( f-i ) Sphere-forming capacity of HK374 spheres treated either with Ehop-016 (Rac GTPase inhibitor) or Rhosin (RhoA-specific inhibitor) or Y27632 (ROCK1/2 inhibitor) or CID44216842 (Cdc42-selective inhibitor) at 500 nM, 1, 5, 10 µM concentrations when combined with radiation and QTP. All experiments have been performed with at least 3 biological independent repeats. p- values were calculated using One-way ANOVA. * p -value < 0.05, ** p -value < 0.01, *** p -value < 0.001, **** p -value < 0.0001.

Article Snippet: To explore the activation of MAPK pathway in vitro , HK374 cells were serum starved and the following day treated with a single dose of 10 Gy in the absence or presence of QTP (10 µM), temozolomide (1 mM; #14163, Cayman Chemical) or Vincristine (250 nM; #HY-N0488, MedChem Express, Monmouth Junction, NJ).

Techniques:

Journal: bioRxiv

Article Title: Activation of the mevalonate pathway in response to anti-cancer treatments drives glioblastoma recurrences through activation of Rac-1

doi: 10.1101/2023.07.23.550205

Figure Lengend Snippet: (a) HK374 cells were treated with 0 or 4 Gy in the presence or absence of QTP (10 µM) and/or atorvastatin (1 µM) for 48 hours. The activated Rac1 was immunoprecipitated by 10 µg PAK-PBD agarose beads from the whole cell lysates and subjected to immunoblotting against Rac1, along with the total proteins. His-tagged Rac1 protein serves as the positive control. (b) The densitometry measurements of activated Rac1/total Rac1 using Image J. (c) Transwell migration assay of HK374 cells pre-treated with 0 or 4 Gy in the presence or absence of QTP (10 µM) and/or atorvastatin (1 µM) for 48 hours. (d) The quantification of migrated cells using Image J. (e) Confocal images of microtubules in HK374 cells treated with 0 or 4 Gy in the presence or absence of QTP (10 µM) and/or atorvastatin (1 µM) for 48 hours. White arrowheads: filopodia. Yellow arrowheads: tunneling nanotubes (TNTs). (f/g) Rac1 knock-down efficiency was evaluated at both mRNA (qRT-PCR) and protein (western blotting) levels at day 2 and day 7 after siRNA transfection. β-actin was used as the loading control and the densitometry measurements of Rac1 using Image J. (h) Clonogenic assay of siCtrl or siRac1 transfected HK374 cells treated with 0 or 4 Gy in the presence or absence of QTP (10 µM) and/or atorvastatin (1 µM) for 7 days. (i) Sphere-forming capacity of siCtrl or siRac1 HK374 spheres treated with 0 or 4 Gy in the presence or absence of QTP (10 µM) and/or atorvastatin (1 µM). All experiments have been performed with at least 3 biological independent repeats. p- values were calculated using One-way ANOVA for b, d ; Unpaired Student’s t-tests for f, g ; Two-way ANOVA for i . * p -value < 0.05, ** p -value < 0.01, *** p -value < 0.001, **** p -value < 0.0001, ns: not significant.

Article Snippet: To explore the activation of MAPK pathway in vitro , HK374 cells were serum starved and the following day treated with a single dose of 10 Gy in the absence or presence of QTP (10 µM), temozolomide (1 mM; #14163, Cayman Chemical) or Vincristine (250 nM; #HY-N0488, MedChem Express, Monmouth Junction, NJ).

Techniques: Immunoprecipitation, Western Blot, Positive Control, Transwell Migration Assay, Knockdown, Quantitative RT-PCR, Transfection, Control, Clonogenic Assay

Journal: Journal of Nephrology

Article Title: Soluble CD59 in peritoneal dialysis: a potential biomarker for peritoneal membrane function

doi: 10.1007/s40620-020-00934-7

Figure Lengend Snippet: Determinants of peritoneal membrane failure or loss of residual kidney function during follow-up

Article Snippet: The ratio was calculated per patient by dividing the PDF level by the plasma level and multiplied by 100%. f The correlation of PDF levels and plasma levels of sCD59 using the Spearman Rank correlation coefficient (r represents the Spearman's rho). sCD59 was measured using an enzyme-linked immunosorbent assay (ELISA; Hycult; HK374-02)

Techniques:

Journal: Journal of Nephrology

Article Title: Soluble CD59 in peritoneal dialysis: a potential biomarker for peritoneal membrane function

doi: 10.1007/s40620-020-00934-7

Figure Lengend Snippet: Local and systemic levels of soluble CD59 in peritoneal dialysis patients. a Violin plot is shown for soluble CD59 (sCD59) levels in the peritoneal dialysis fluid (PDF). The width of the shape indicates the probability density of patients with a given result. The lines represent the median (blue horizontal line), interquartile range (25th–75th percentile, black horizontal lines). b Violin plot is shown for soluble C5b-9 (sC5b-9) levels in the PDF. sC5b-9 was detectable in the peritoneal dialysate of all patients (n = 48). c The correlation of PDF levels of sCD59 with sC5b-9 using the Spearman Rank correlation coefficient. The dashed lines show the 95% confidence interval for the regression line (blue). d sCD59 plasma levels were determined in; healthy controls (n = 14), non-dialysis dependent chronic kidney disease (CKD) patients (n = 15), hemodialysis (HD) patients prior to dialysis (n = 41), PD patients (n = 48). Average age was 56 ± 4 years in healthy controls, 77 ± 11 years in CKD patients, and 66 ± 16 years in HD patients, and 64%, 60% and 70% were male, respectively. Data are presented as median plus interquartile range and were analyzed by Kruskal Wallis test with an option for multiple comparisons (*** P < 0.001). e Violin plot for the PDF—plasma ratio of sCD59 in dialysis patients (n = 48). The ratio was calculated per patient by dividing the PDF level by the plasma level and multiplied by 100%. f The correlation of PDF levels and plasma levels of sCD59 using the Spearman Rank correlation coefficient (r represents the Spearman's rho). sCD59 was measured using an enzyme-linked immunosorbent assay (ELISA; Hycult; HK374-02)

Article Snippet: The ratio was calculated per patient by dividing the PDF level by the plasma level and multiplied by 100%. f The correlation of PDF levels and plasma levels of sCD59 using the Spearman Rank correlation coefficient (r represents the Spearman's rho). sCD59 was measured using an enzyme-linked immunosorbent assay (ELISA; Hycult; HK374-02)

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of Nephrology

Article Title: Soluble CD59 in peritoneal dialysis: a potential biomarker for peritoneal membrane function

doi: 10.1007/s40620-020-00934-7

Figure Lengend Snippet: The relationship between soluble CD59 and diafiltration. a The correlation between residual renal function and plasma sCD59 and b the correlation between baseline transport status and the PDF/plasma ratio of sCD59 using the Spearman Rank correlation coefficient (r represents the Spearman's rho). The dashed lines show the 95% confidence interval for the regression line (blue). A significant correlation was found between the plasma sCD59 levels and residual renal function, and sCD59 ratio and transport status. c HD significantly reduced plasma sCD59 levels (*** P < 0.001). d Violin plot for the pre-HD/post-HD ratio of plasma sCD59 levels in dialysis patients (n = 25). The ratio was calculated per patient by dividing the pre-HD level by the post-HD level and multiplied by 100%. The width of the shape indicates the probability density of patients with a given result. The lines represent the median (blue horizontal line), interquartile range (25th–75th percentile, black horizontal lines)

Article Snippet: The ratio was calculated per patient by dividing the PDF level by the plasma level and multiplied by 100%. f The correlation of PDF levels and plasma levels of sCD59 using the Spearman Rank correlation coefficient (r represents the Spearman's rho). sCD59 was measured using an enzyme-linked immunosorbent assay (ELISA; Hycult; HK374-02)

Techniques: Diafiltration Assay

Journal: Journal of Nephrology

Article Title: Soluble CD59 in peritoneal dialysis: a potential biomarker for peritoneal membrane function

doi: 10.1007/s40620-020-00934-7

Figure Lengend Snippet: Soluble CD59 as a biomarker for transport status, membrane failure and loss of residual renal function during follow up. a The difference in peritoneal dialysis fluid (PDF) levels of soluble CD59 (sCD59) were analyzed between PD patient groups with low (D/P creatinine < 0.65) and high (D/P creatinine > 0.65) transport status after 1 year of follow-up. b The correlation between PDF sCD59 levels and transport status after 1 year using the Spearman Rank correlation coefficient (r represents the Spearman's rho). The dashed lines show the 95% confidence interval for the regression line (blue). c The difference in PDF sCD59 levels between PD patients with and without peritoneal membrane failure (PMF) during follow-up. Data are presented as median plus interquartile range and were analyzed by Mann–Whitney test (** P < 0.01). Cumulative event-free survival for peritoneal membrane failure among PD patients with low and high sCD59 levels in the PDF ( d ) or in the plasma ( e ). Cumulative event-free survival for loss of residual renal function among PD patients with low and high plasma levels of sCD59 levels ( f ). Log-rank test was used to compare the incidence of PMF and loss of residual renal function between the groups. High sCD59 levels in the PDF (> 38.6 ng/mL) and plasma (> 219 ng/mL) are both associated with lower survival of the peritoneal membrane. High plasma CD59 (> 219 ng/mL) was significantly associated with loss of residual renal function

Article Snippet: The ratio was calculated per patient by dividing the PDF level by the plasma level and multiplied by 100%. f The correlation of PDF levels and plasma levels of sCD59 using the Spearman Rank correlation coefficient (r represents the Spearman's rho). sCD59 was measured using an enzyme-linked immunosorbent assay (ELISA; Hycult; HK374-02)

Techniques: Biomarker Assay, MANN-WHITNEY