phage braf v600e  (Addgene inc)

Journal: Cell reports

Article Title: ERK1/2 interaction with DHPS regulates eIF5A deoxyhypusination independently of ERK kinase activity

doi: 10.1016/j.celrep.2024.114831

Figure Lengend Snippet: (A) Western blotting of total lysates of HEK293-ΔRaf:ER cells subjected to tamoxifen-induced ΔRaf:ER activation for indicated periods to determine time-dependent MEK/ERK effects on DHPS and eIF5A. eIF5A D/H collectively indicates deoxyhypusinated eIF5A and hypusinated eIF5A. Densitometry (right) of western blot signals at 48 h was normalized to β-actin. (B) Western blotting of total lysates of HEK293 cells infected with lentiviral pHAGE expressing N-terminal HA-tagged wild-type MEK1 (MEK1wt) and constitutively active MEK1 (MEK1ca) for indicated periods to determine time-dependent MEK/ERK effects on DHPS and eIF5A. Densitometry (right) of western blot signals was normalized to β-actin. (C) Western blotting of total lysates of A375 cells treated with 1 μM PLX4032 or 100 nM AZD6244 for 48 h to determine how BRAF or MEK1/2 inhibition affects DHPS and eIF5A in BRAF V600E tumor cells. An equal volume of DMSO was used as the vehicle control. Densitometry (right) of western blot signals was normalized to β-actin. See also for additional data. (D) Western blotting of total lysates of HEK293 cells infected with lentiviral pLL3.7 expressing short hairpin RNA (shRNA) targeting ERK1 (shERK1) or ERK2 (shERK2) for 2 days and then switched to fresh media for indicated periods to determine ERK knockdown effects on DHPS and eIF5A. Densitometry (right) of western blot signals was normalized to β-actin. See also and for additional data. (E) Western blotting of total lysates of HEK293-ΔRaf:ER cells subjected to 24 h tamoxifen-induced ΔRaf:ER activation in the presence or absence of ERK1/2 inhibitors acting through different mechanisms. SCH-772984 inhibits the ERK1/2 activation loop and catalytic site. LY-5214996 inhibits the catalytic site only. Data are mean ± SD of biological triplicates, where * p < 0.05, ** p < 0.005, and *** p < 0.001 (by two-tailed Student’s t test).

Article Snippet: Construction of the lentiviral pHAGE-ΔRaf:ER, pHAGE-BRAF V600E , pHAGE-MEK1-R4F (ΔN3/S218E/S222D, constitutively active MEK1), pLL3.7-shERK1, and pLL3.7-shERK2 was previously described., C-terminally HA-tagged DHPS in pHAGE-GFP was generated by ligating the full length DHPS cDNA from pCMV3-ORF-HA (Sino Biologicals) to the NheI/XhoI sites in pHAGE-GFP.

Techniques: Western Blot, Activation Assay, Infection, Expressing, Inhibition, Control, shRNA, Knockdown, Two Tailed Test

Journal: Cell reports

Article Title: ERK1/2 interaction with DHPS regulates eIF5A deoxyhypusination independently of ERK kinase activity

doi: 10.1016/j.celrep.2024.114831

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Construction of the lentiviral pHAGE-ΔRaf:ER, pHAGE-BRAF V600E , pHAGE-MEK1-R4F (ΔN3/S218E/S222D, constitutively active MEK1), pLL3.7-shERK1, and pLL3.7-shERK2 was previously described., C-terminally HA-tagged DHPS in pHAGE-GFP was generated by ligating the full length DHPS cDNA from pCMV3-ORF-HA (Sino Biologicals) to the NheI/XhoI sites in pHAGE-GFP.

Techniques: Virus, Recombinant, Labeling, Transfection, Expressing, Software

Journal: Neural Regeneration Research

Article Title: Oncogenic BRAF V600E induces microglial proliferation through extracellular signal-regulated kinase and neuronal death through c-Jun N-terminal kinase

doi: 10.4103/1673-5374.361516

Figure Lengend Snippet: BRAF V600E expression leads to neuronal cell death and glial cell proliferation in primary mouse cortical mixed culture. Primary cortex mix cultures were prepared from C57BL/6J embryos. Cells were cultured in NB-A for 5 days, then transduced with viral vector, or BRAF WT , or BRAF V600E for 24 hours and then cultured in NB-A medium for 96 hours. (A) BRAF immunoblot and quantified BRAF expression level normalized to GAPDH. (B) Immunostaining for MAP2 and Iba1, and MAP2 + and Iba1 + cell counts in cultures transduced with lentiviral BRAF vectors. MAP2 (red, neurons), Iba1 (green, microglia), DAPI (blue, nuclei). (C) BRAF immunoblot and quantified BRAF expression level normalized to GAPDH. (D) Immunostaining for MAP2 and Iba1, and MAP2 + and Iba1 + cell counts in cultures transduced with retroviral (RV) BRAF vectors. MAP2 (red, neurons), Iba1 (green, microglia), DAPI (blue, nuclei). (E) Immunostaining for MAP2 and GFAP, and MAP2 + and GFAP + cell counting in cultures transduced with lentiviral BRAF vectors. MAP2 (green, neurons), GFAP (red, astrocytes), DAPI (blue, nuclei). (F) Immunostaining for MAP2 and GFAP, and MAP2 + cells and GFAP + cell counting in cultures transduced with RV BRAF vectors. The nuclei were counterstained by DAPI. MAP2 (green, neurons), GFAP (red, astrocytes), DAPI (blue, nuclei). Bars: 100 μm. ** P < 0.01, *** P < 0.001 (one-way analysis of variance and Tukey’s post hoc test). All experiments were repeated at least three times with at least three replicates under each condition. DAPI: 4′,6-Diamidino-2-phenylindole; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; MAP2: microtubule-associated protein 2; NB-A: neurobasal media.

Article Snippet: Constructs used were: pMD2.G (Addgene, Cat# 12259, RRID: Addgene_12259), psPAX2 (Addgene, Cat# 12260, RRID: Addgene_12260), pHAGE- BRAF V600E plasmid (Addgene, Cat# 116204, RRID: Addgene_116204), pHAGE- BRAF WT plasmid (Addgene, Cat# 116719, RRID: Addgene_116719), pBabe-Puro- BRAF V600E plasmid (Addgene, Cat# 15269, RRID: Addgene_15269), gag/pol-Retroviral plasmid (Addgene, Cat# 14887, RRID: Addgene_14887), Control siRNA (Cell Signaling Technology, Boston, MA, USA, Cat# 6568S), SAPK/JNK siRNA (Cell Signaling Technology, Cat# 6232S), ERK1 siRNA (Cell Signaling Technology, Cat# 6436S), ERK2 siRNA (Cell Signaling Technology, Cat# 6578S).

Techniques: Expressing, Cell Culture, Transduction, Plasmid Preparation, Western Blot, Immunostaining, Retroviral, Cell Counting

Journal: Neural Regeneration Research

Article Title: Oncogenic BRAF V600E induces microglial proliferation through extracellular signal-regulated kinase and neuronal death through c-Jun N-terminal kinase

doi: 10.4103/1673-5374.361516

Figure Lengend Snippet: BRAF V600E expression in astrocytes promotes proliferation. Primary astrocytes were prepared from C57BL/6J embryos. Cells were transduced with lentiviral vector, or BRAF WT , or BRAF V600E for 24 hours and then cultured in DMEM/F-12 medium for 96 hours. (A) Immunoblotting for BRAF and related signaling proteins and quantified expression levels normalized to GAPDH. (B) Flow cytometry analysis of cell cycle. (C) MTS cell viability assay at 48, 72, 96 and 120 hours after viral transduction. (D) Immunostaining for GFAP and Ki67, GFAP + cell counting, and % of Ki67 + astrocytes (scale bar: 100 μm). GFAP (red, astrocytes), DAPI (blue, nuclei), Ki-67 (green, proliferative cells). (E, F) qPCR analysis of inflammatory and antioxidant markers normalized to GAPDH. ** P < 0.01, *** P < 0.001 (one-way analysis of variance and Tukey’s post hoc test). All experiments were repeated at least three times with at least three replicates within each condition. DAPI: 4′,6-Diamidino-2-phenylindole; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary acidic protein; qPCR: quantitative polymerase chain reaction.

Article Snippet: Constructs used were: pMD2.G (Addgene, Cat# 12259, RRID: Addgene_12259), psPAX2 (Addgene, Cat# 12260, RRID: Addgene_12260), pHAGE- BRAF V600E plasmid (Addgene, Cat# 116204, RRID: Addgene_116204), pHAGE- BRAF WT plasmid (Addgene, Cat# 116719, RRID: Addgene_116719), pBabe-Puro- BRAF V600E plasmid (Addgene, Cat# 15269, RRID: Addgene_15269), gag/pol-Retroviral plasmid (Addgene, Cat# 14887, RRID: Addgene_14887), Control siRNA (Cell Signaling Technology, Boston, MA, USA, Cat# 6568S), SAPK/JNK siRNA (Cell Signaling Technology, Cat# 6232S), ERK1 siRNA (Cell Signaling Technology, Cat# 6436S), ERK2 siRNA (Cell Signaling Technology, Cat# 6578S).

Techniques: Expressing, Transduction, Plasmid Preparation, Cell Culture, Western Blot, Flow Cytometry, Viability Assay, Immunostaining, Cell Counting, Real-time Polymerase Chain Reaction

Journal: Neural Regeneration Research

Article Title: Oncogenic BRAF V600E induces microglial proliferation through extracellular signal-regulated kinase and neuronal death through c-Jun N-terminal kinase

doi: 10.4103/1673-5374.361516

Figure Lengend Snippet: BRAF V600E expression in microglia induces cell proliferation and activation through ERK. Primary microglia cells were prepared from C57BL/6J embryos. Cells were transduced with lentiviral vector, BRAF WT and BRAF V600E for 24 hours and then cultured in DMEM/F12 for 96 hours. (A) Immunoblotting for BRAF and related signaling proteins and quantified expression levels normalized to GAPDH. (B, C) Cells were transfected with control-siRNA, or si-JNK, or si-ERK for 24 hours before transduction with BRAF viral vectors. Immunoblotting for ERK and JNK 48 hours after viral transduction and quantified expression levels normalized to GAPDH. (D) Cells were transfected with control-siRNA, or si-JNK, or si-ERK for 24 hours before transduction with BRAF viral vectors. Immunoblotting for BRAF-related signaling proteins and quantified expression levels normalized to GAPDH. (E, F) Immunostaining for Iba1 and Ki67, Iba1 + cell counts, and percentage of Ki67 + microglia (scale bar: 100 μm), and quantitative morphological analyses (percentage of ameboid-like microglia cells, length, area, length to area ratio in cells without and with siRNA transfection (scale bar: 50 μm). Iba1 (green, astrocytes), DAPI (blue, nuclei), Ki67 (red, proliferative cells). (G, H) Flow cytometry analysis of cell cycle. (I) MTS cell viability assay at 48, 72, 96 and 120 hours following viral transduction. (J) NO release in culture media by Griess reaction. (K) qPCR analysis of inflammatory and antioxidant markers in cells normalized to GAPDH. (L) IL-1β, IL-6 and TNF-α levels in culture medium measured by ELISA. Data are represented as mean ± SEM, n = 9. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance and Tukey’s post hoc test). All experiments were repeated at least three times with at least three replicates under each condition. ERK: Extracellular signal-regulated kinase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; JNK: c-Jun N-terminal kinase; NO: nitric oxide; qPCR: quantitative polymerase chain reaction.

Article Snippet: Constructs used were: pMD2.G (Addgene, Cat# 12259, RRID: Addgene_12259), psPAX2 (Addgene, Cat# 12260, RRID: Addgene_12260), pHAGE- BRAF V600E plasmid (Addgene, Cat# 116204, RRID: Addgene_116204), pHAGE- BRAF WT plasmid (Addgene, Cat# 116719, RRID: Addgene_116719), pBabe-Puro- BRAF V600E plasmid (Addgene, Cat# 15269, RRID: Addgene_15269), gag/pol-Retroviral plasmid (Addgene, Cat# 14887, RRID: Addgene_14887), Control siRNA (Cell Signaling Technology, Boston, MA, USA, Cat# 6568S), SAPK/JNK siRNA (Cell Signaling Technology, Cat# 6232S), ERK1 siRNA (Cell Signaling Technology, Cat# 6436S), ERK2 siRNA (Cell Signaling Technology, Cat# 6578S).

Techniques: Expressing, Activation Assay, Transduction, Plasmid Preparation, Cell Culture, Western Blot, Transfection, Control, Immunostaining, Flow Cytometry, Viability Assay, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Neural Regeneration Research

Article Title: Oncogenic BRAF V600E induces microglial proliferation through extracellular signal-regulated kinase and neuronal death through c-Jun N-terminal kinase

doi: 10.4103/1673-5374.361516

Figure Lengend Snippet: Conditioned medium from BRAF V600E -expressing microglial cells but not astrocytes induces neuronal cell death. Primary cortex neurons were prepared from C57BL/6J embryos and cultured for 5 days, and the original medium was then replaced with conditional medium for 72 hours. (A, B) Immunostaining for MAP2 (scale bar: 50 μm) and MAP2 + cell counting. (C) LDH release in neurons treated with conditioned medium from primary astrocytes transduced with lentiviral BRAF vectors. (D–F) Immunostaining for MAP2 (scale bar: 50 μm; D) and MAP2 + cell counting in neurons treated with conditioned medium from primary microglia transduced with lentiviral BRAF vectors with and without siRNA transfection (E, F). (G, H) LDH release from neurons treated with conditioned medium from primary microglia transduced with lentiviral BRAF vectors with and without siRNA transfection. * P < 0.05, *** P < 0.001 (one-way analysis of variance and Tukey’s post hoc test). All experiments were repeated at least three times with at least three replicates within each condition. MAP2 (green, neurons), DAPI (blue, nuclei) in A and D. DAPI: 4′,6-Diamidino-2-phenylindole; LDH: lactate dehydrogenase; MAP2: microtubule-associated protein 2.

Article Snippet: Constructs used were: pMD2.G (Addgene, Cat# 12259, RRID: Addgene_12259), psPAX2 (Addgene, Cat# 12260, RRID: Addgene_12260), pHAGE- BRAF V600E plasmid (Addgene, Cat# 116204, RRID: Addgene_116204), pHAGE- BRAF WT plasmid (Addgene, Cat# 116719, RRID: Addgene_116719), pBabe-Puro- BRAF V600E plasmid (Addgene, Cat# 15269, RRID: Addgene_15269), gag/pol-Retroviral plasmid (Addgene, Cat# 14887, RRID: Addgene_14887), Control siRNA (Cell Signaling Technology, Boston, MA, USA, Cat# 6568S), SAPK/JNK siRNA (Cell Signaling Technology, Cat# 6232S), ERK1 siRNA (Cell Signaling Technology, Cat# 6436S), ERK2 siRNA (Cell Signaling Technology, Cat# 6578S).

Techniques: Expressing, Cell Culture, Immunostaining, Cell Counting, Transduction, Transfection

Journal: Neural Regeneration Research

Article Title: Oncogenic BRAF V600E induces microglial proliferation through extracellular signal-regulated kinase and neuronal death through c-Jun N-terminal kinase

doi: 10.4103/1673-5374.361516

Figure Lengend Snippet: BRAF V600E expression in neurons promotes cell death through the JNK pathway. Primary cortex neurons were prepared from C57BL/6J embryos. Cells were cultured for 5 days, transduced with lentiviral vector, BRAF WT and BRAF V600E for 24 hours, and then cultured in NB-A for 96 hours. (A) Immunoblotting for BRAF and related signaling proteins and quantified expression levels normalized to GAPDH. (B) qPCR analysis c-Jun, Bax, Bcl-2, p53, Fasl and TNF-α normalized to GAPDH. (C–E) Immunostaining for MAP2 (scale bar: 50 μm), MAP2 + cell counting, and LDH release. (F) Primary cortex neurons were transfected with control-siRNA or si-JNK for 24 hours before transduction with BRAF viral vectors. Immunoblotting for BRAF and related signaling proteins and quantified expression levels normalized to GAPDH. (G) qPCR analysis of c-Jun, Bax, Bcl-2, p53, Fasl and TNF-α normalized to GAPDH. (H) Immunostaining for MAP2 (sale bar: 50 μm), (I) MAP2 + cell counting, (J) and LDH release. Primary cortex neurons were transfected with control-siRNA or si-ERK for 24 hours before transduction with BRAF viral vectors. (K) Immunoblotting for BRAF and related signaling proteins and quantified expression levels normalized to GAPDH. (L) qPCR analysis of c-Jun, Bax, Bcl-2, p53, Fasl and TNF-α normalized to GAPDH. (M–O) Immunostaining for MAP2 (scale bar: 50 μm), MAP2 + cell counting, and LDH release. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance and Tukey’s post hoc test). All experiments were repeated at least three times with at least three replicates within each condition. MAP2 (green, neurons), DAPI (blue, nuclei) in C, H, and M. DAPI: 4′,6-Diamidino-2-phenylindole; ERK: extracellular signal-regulated kinase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; JNK: c-Jun N-terminal kinase; LDH: lactate dehydrogenase; MAP2: microtubule-associated protein 2; NB-A: neurobasal media; qPCR: quantitative polymerase chain reaction; TNF-α: tumor necrosis factor-alpha; WT: wide type.

Article Snippet: Constructs used were: pMD2.G (Addgene, Cat# 12259, RRID: Addgene_12259), psPAX2 (Addgene, Cat# 12260, RRID: Addgene_12260), pHAGE- BRAF V600E plasmid (Addgene, Cat# 116204, RRID: Addgene_116204), pHAGE- BRAF WT plasmid (Addgene, Cat# 116719, RRID: Addgene_116719), pBabe-Puro- BRAF V600E plasmid (Addgene, Cat# 15269, RRID: Addgene_15269), gag/pol-Retroviral plasmid (Addgene, Cat# 14887, RRID: Addgene_14887), Control siRNA (Cell Signaling Technology, Boston, MA, USA, Cat# 6568S), SAPK/JNK siRNA (Cell Signaling Technology, Cat# 6232S), ERK1 siRNA (Cell Signaling Technology, Cat# 6436S), ERK2 siRNA (Cell Signaling Technology, Cat# 6578S).

Techniques: Expressing, Cell Culture, Transduction, Plasmid Preparation, Western Blot, Immunostaining, Cell Counting, Transfection, Control, Real-time Polymerase Chain Reaction