plasmid coding for eif2a variants wt, s51a, and s51d heif2a variants in pcdna3.cd2 expression vectors  (Addgene inc)

Journal: Frontiers in oncology

Article Title: Pediatric glioblastoma cells are sensitive to drugs that inhibit eIF2α dephosphorylation and its phosphomimetic S51D variant.

doi: 10.3389/fonc.2022.959133

Figure Lengend Snippet: FIGURE 1 Combined treatment of vorinostat and PARPis enhanced the decrease in PED-GBM survival: SU-DIPG-VI cells (A, B) and SU-DIPG-IV cells (C, D) were plated in triplicates at a density of 11,550 or 3,300 cells/cm2, respectively. Drugs were added 24 h post-plating. KNS-42 were plated either for clonogenic assay (E) or for survival assay (F) at a density of 55 or 6,600 cells/cm2, respectively. Values are mean survival (%) ± S.D relative to control. Differences among all experimental groups, as well as between each experimental group and control, were significant - *p< 0.5, **p< 0.005. (G–I). Cells were processed for Western blot analysis of treatment-induced changes in the ratio of peIF2a/eIF2a. Numbers at the bottom of the autoradiograms indicate changes of peIF2a/eIF2a and differences in loaded proteins (Ponceau) relative to control. (J, K). SU-DIPG VI and HAs cells were plated in triplicates at the same density (3,300 cells/cm2) and treated with drugs 24 h post-plating. Values are the mean number of cell division or cell death (%) ± S.D relative to control. Differences between treated and untreated experimental groups, as well as between SU-DIPG-VI and HAs, were significant *p< 0.05, **p< 0.005. V—vorinostat, Ola—Olaparib, Vel—veliparib, Nir—niraparib. The experiments were reproduced at least twice with similar results.

Article Snippet: Frontiers in Oncology 04 Transfection of cells with plasmid coding for eIF2a variants WT, S51A, and S51D heIF2a variants in pcDNA3.CD2 expression vectors were obtained from Addgene (Cambridge, MA).

Techniques: Clonogenic Assay, Clonogenic Cell Survival Assay, Control, Western Blot

Journal: Frontiers in oncology

Article Title: Pediatric glioblastoma cells are sensitive to drugs that inhibit eIF2α dephosphorylation and its phosphomimetic S51D variant.

doi: 10.3389/fonc.2022.959133

Figure Lengend Snippet: FIGURE 2 Salubrinal and raphin-1 increase P-eIF2a: (A–C). Cells were processed for Western blot analysis of treatment-induced changes in the ratio of peIF2a/eIF2a at the indicated time. Numbers at the bottom of the autoradiograms indicate changes of peIF2a/eIF2a and differences in loaded proteins (Ponceau) relative to control. Sal—salubrinal, R1—raphin-1. The experiments were reproduced at least twice with similar results.

Article Snippet: Frontiers in Oncology 04 Transfection of cells with plasmid coding for eIF2a variants WT, S51A, and S51D heIF2a variants in pcDNA3.CD2 expression vectors were obtained from Addgene (Cambridge, MA).

Techniques: Western Blot, Control

Journal: Frontiers in oncology

Article Title: Pediatric glioblastoma cells are sensitive to drugs that inhibit eIF2α dephosphorylation and its phosphomimetic S51D variant.

doi: 10.3389/fonc.2022.959133

Figure Lengend Snippet: FIGURE 3 Salubrinal and raphin-1, as well as S51D-eIF2a, decreased cell survival and affected the eIF2Be level and phosphorylation: (A, B). SU-DIPG-VI and SU-DIPG-IV cells were plated in triplicates at a density of 11,550 or 3,300 cells/cm2, respectively, and treated 24 h post-plating. (C). KNS-42 cells were plated in triplicates for clonogenic survival assay and treated 24 h post-plating. Values (A–C) are mean survival (%) ± S.D relative to control. Differences among all experimental groups, as well as between each experimental group and control, were significant—*p< 0.5, **p< 0.005. (D). A representative image of SU-DIPG-VI neurospheres 18 days post-treatment with salubrinal and raphin-1. Bar, 400 mm. (E). SU-DIPG VI and HAs cells were plated in triplicates at the same density (3,300 cells/cm2) and treated 24 h post-plating. Values are mean number of cell division or cell death (%) ± S.D relative to control. Differences between treated and untreated experimental groups, as well as between SU- DIPG-VI and HAs, were significant—*p< 0.05, **p< 0.005. (F). SU-DIPG-VI cells were processed for Western blot analysis of treatment-induced changes in the ratios of peIF2a/eIF2a and peIF2Be/eIF2Be and in the cellular level of eIF2Be. Numbers at the bottom of the autoradiograms indicate changes of peIF2a/eIF2a, peIF2Be/eIF2Be, and eIF2Be and differences in loaded proteins (Ponceau) relative to control. (G). KNS-42 cells were plated in duplicates at a density of 13,200 cells/cm2 and transiently transfected with 1 mg/ml of plasmids expressing HA-eIF2a (HA-WT) or HA-S51A-eIF2a (HA-SA) or with HA-S51D-eIF2a (HA-SD) 24 h post-plating. Values are mean survival (%) ± S.D relative to control. Differences between survival of HA-SD to the HA-WT and HA-SA were significant—**p< 0.005. (H). Cells were transiently transfected as described in G and 12 h later processed for Western blot analysis of HA-tag expression. Numbers at the bottom of the autoradiograms indicate differences in loaded proteins (Ponceau) relative to HA-WT. Sal—salubrinal, R1—raphin-1. The experiments were reproduced at least twice with similar results.

Article Snippet: Frontiers in Oncology 04 Transfection of cells with plasmid coding for eIF2a variants WT, S51A, and S51D heIF2a variants in pcDNA3.CD2 expression vectors were obtained from Addgene (Cambridge, MA).

Techniques: Phospho-proteomics, Clonogenic Cell Survival Assay, Control, Western Blot, Transfection, Expressing

Journal: Frontiers in oncology

Article Title: Pediatric glioblastoma cells are sensitive to drugs that inhibit eIF2α dephosphorylation and its phosphomimetic S51D variant.

doi: 10.3389/fonc.2022.959133

Figure Lengend Snippet: FIGURE 5 Combination of PARPis and salubrinal or raphin-1 enhanced the decrease in cell survival. (A–C) Cells were processed for Western blot analysis of treatment-induced changes in the ratio of peIF2a/eIF2a. Numbers at the bottom of the autoradiograms indicate changes of peIF2a/eIF2a and differences in loaded proteins (Ponceau) relative to control. (D, E). SU-DIPG-VI cells were plated at a density of 11,550 cells/cm2, treated with drugs either 24 h (D) or 120 h (E) post-plating and incubated for additional 7 days. (F). KNS-42 cells were plated in triplicates for clonogenic assay at a density of 55 cells/cm2 and treated with drugs 24 h post-plating. Values are mean survival or cell death (%) ± S.D relative to control. Differences among all experimental groups, as well as between each experimental group and control, were significant—*p< 0.05, **p< 0.005 (D–F). (G, H). SU-DIPG VI and HAs cells were plated in triplicates at a density of 3,300 cells/cm2 and treated with drugs 24 h post-plating. Values are mean number of cell division or cell death (%) ± S.D relative to control. Differences between treated and untreated experimental groups, as well as between SU-DIPG-VI and HAs, were significant—*p< 0.05, **p< 0.005. (I). KNS-42 cells were plated in duplicates at a density of 13,200 cells/cm2 and transiently transfected with 1 mg/ml of plasmids expressing HA-eIF2a (HA-WT) or HA-S51A-eIF2a (HA-SA), or with HA- S51D-eIF2a (HA-SD) 24 h post-plating. Twenty-four hours later, cells were treated with niraparib for 6 days. Values are mean survival (%) ± S.D relative to control. Differences between survival of HA-SD with or without niraparib to either treated or untreated HA-SA or HA-WT were significant—*p< 0.05, **p< 0.005. Sal—salubrinal, R1—raphin-1, Ola—olaparib, Nir—niraparib. The experiments were reproduced at least twice with similar results.

Article Snippet: Frontiers in Oncology 04 Transfection of cells with plasmid coding for eIF2a variants WT, S51A, and S51D heIF2a variants in pcDNA3.CD2 expression vectors were obtained from Addgene (Cambridge, MA).

Techniques: Western Blot, Control, Incubation, Clonogenic Assay, Transfection, Expressing

Journal: Frontiers in oncology

Article Title: Pediatric glioblastoma cells are sensitive to drugs that inhibit eIF2α dephosphorylation and its phosphomimetic S51D variant.

doi: 10.3389/fonc.2022.959133

Figure Lengend Snippet: FIGURE 6 Combination of radiation with salubrinal or raphin-1 enhanced decrease in cell survival: (A). SU-DIPG-VI cells were irradiated and treated with drugs 24 h post-plating and incubated for additional 48 h before processing for Western blot analysis of treatment-induced changes in the ratio of peIF2a/eIF2a. Numbers at the bottom of the autoradiograms indicate changes of peIF2a/eIF2a and differences in loaded proteins (Ponceau) relative to control. (B). SU-DIPG-VI cells were plated in triplicates at a density of 11,500 cells/cm2 and 24 h later were irradiated and treated with salubrinal. Cells were counted 7 days following treatment initiation. Values are mean survival (%) ± S.D relative to control. Differences among all experimental groups, as well as between each experimental group and control, were significant—*p< 0.05. (C). SU-DIPG VI and HA cells were plated in triplicates at a density of 3,300 cells/cm2 and treated with drugs and radiation 24 h post-plating. Values are mean number of cell division or cell death (%) ± S.D relative to control. Differences among all experimental groups as well as between each experimental group and control were significant for SU-DIPG-VI. When indicated, significance was observed between treated and control untreated HAs, but not among all experimental groups—*p< 0.05, **p< 0.005. Gy—gray, Sal—salubrinal, R1—Raphin-1. The experiments were reproduced once with similar results.

Article Snippet: Frontiers in Oncology 04 Transfection of cells with plasmid coding for eIF2a variants WT, S51A, and S51D heIF2a variants in pcDNA3.CD2 expression vectors were obtained from Addgene (Cambridge, MA).

Techniques: Irradiation, Incubation, Western Blot, Control