Journal: Cell reports

Article Title: Driving Neuronal Differentiation through Reversal of an ERK1/2-miR-124-SOX9 Axis Abrogates Glioblastoma Aggressiveness.

doi: 10.1016/j.celrep.2019.07.071

Figure Lengend Snippet: Figure 3. miR-124 Is Expressed in an ERK1/2-Dependent Manner in Glioma Cells (A) Selected clusters of miRNA associated with neurogenesis from NanoString miRNA profiling in adult WT mouse brain (n = 3) versus mouse G-Ras astrocytomas (tumor, n = 4), including downregulated miR-124 in tumors (*p < 0.05; ANOVA Bonferroni’s post hoc test). See also Table S4. (B) Isolated V-SVZ mouse neurospheres form P4 N-TVA mice infected with conditioned media from DF1 cells producing R-CAS-K-RasG12D retrovirus to transduce oncogenic K-RasG12D. (C) NanoString miRNA profiling showing examples of neurogenesis-related miRNAs in V-SVZ N-TVA mouse neurospheres that were regulated in an ERK1/2- dependent manner with or without oncogenic K-RasG12D expression and incubated with PD0325901 for (1 mM) for 3 days. See also Table S5. (D) TCGA dataset showing differential expression of miR-124 in transcriptionally defined human GBMs (n = 158, ***p < 0.001, one-way ANOVA with Tukey’s post hoc test). CL, classical; MES, mesenchymal; NL, neural; PN, proneural. (E) Semiquantitative assessment of miR-124 levels based on PCR-based miRNA array profiling with or without PD0325901 (1 mM) incubation of patient-derived proneural GBM5 or classical GBM6 tumorsphere cultures after 5 days. (F) Heatmap showing semiquantitative analysis of miRNAs with or without PD0325901 (1 mM) in GBM5 or GBM6 tumorsphere cultures after 5 days. See also Table S6. (G) Quantitative real-time PCR (dCt ± SD, n = 3, *p < 0.05; ANOVA Tukey’s post hoc test) of miR-9 expression following 5 days with or without PD0325901 (1 mM) in proneural GBM5 and mesenchymal GBM43 tumorsphere cultures. (H) Quantitative real-time PCR (dCt ± SD, n = 3, ***p < 0.001; ANOVA Tukey’s post hoc test) of miR-124 expression following 5 days with or without PD0325901 (1 mM) in GBM5 and GBM43 tumorsphere cultures.

Article Snippet: MEK inhibition in vivo PD0325901 (Tocris Bioscience) was solubilized in vehicle (0.5% hydroxypropylmethyl-cellulose/0.2% Tween80).

Techniques: Isolation, Infection, Transduction, Expressing, Incubation, Quantitative Proteomics, Derivative Assay, Real-time Polymerase Chain Reaction

Journal: Cell reports

Article Title: Driving Neuronal Differentiation through Reversal of an ERK1/2-miR-124-SOX9 Axis Abrogates Glioblastoma Aggressiveness.

doi: 10.1016/j.celrep.2019.07.071

Figure Lengend Snippet: Figure 5. PD0325901-Induced Neuronal Differentiation Requires miR-124 Induction and SOX9 Depletion in EGFRvIII-Driven GBM Models (A) Design of mouse GBM model based on V-SVZ neurospheres isolated from postnatal Ink4a/Arf null mice, lentivirally transduced with hEGFRvIII (vIII cells), mCherry, and firefly luciferase (LUC), followed by allografting into the frontal cortex of recipient adult FVBN mice. (B) Mice developing GBMs were administered vehicle or the MEK inhibitor PD0325901 (PD; 5 mg/kg/day) for 10 days. (C and D) Expression of SOX9 and cleaved caspase3 in EGFRvIII-driven mouse GBMs after 10 days’ treatment with vehicle (C) or PD0325901 (D). Scale bar: 40 mm. (E) Bioluminescence imaging as readout of tumor growth in mice following treatment with vehicle (F and H) or PD0325901 (G and I) (means ± SEMs, n = 5, **p < 0.01; Student’s t test). (F–I) Expression of post-mitotic neuron marker Map2ab and immature neuron marker Tuj1 following treatment with vehicle or PD0325901. Scale bar: 20 mm. (J) Lentiviral transduction with hSOX9 (vIII.hSOX9) or a lentiviral miR-124 sponge (vIII.124sp) in vIII cells. (K) Design of lentiviral sponge vector to stably inhibit miR-124 function. (L and M) Downregulation of SOX9 and neuronal differentiation (Tuj1) after 6 days in untreated (control) vIII cells (L) or following incubation with PD0325901 (M) (PD, 1 mM). Scale bar: 50 mm. (N) Quantification of Tuj1 expression following PD0325901 (1 mM) in vIII, vIII.124sp, and vIII.SOX9 cultures after 6 days (means ± SEMs, n = 4, **p < 0.01; Student’s t test). (O) Immunoblotting for WT EGFR and EGFRvIII protein in human GBM cultures. (P and Q) Labeling of EGFRvIII mutated GBM6 cells with antibodies against SOX9 and Tuj1 after 6 days following incubation from DMSO (control) (P) or PD0325901 (1 mM) (O). (R) Quantification (means ± SEMs, n = 3, ***p < 0.001; Student’s test) of GBM6 and GBM6.124sp cells following incubation with PD0325901 (1 mM) for 6 days. See also Figure S4.

Article Snippet: MEK inhibition in vivo PD0325901 (Tocris Bioscience) was solubilized in vehicle (0.5% hydroxypropylmethyl-cellulose/0.2% Tween80).

Techniques: Isolation, Transduction, Luciferase, Expressing, Imaging, Marker, Plasmid Preparation, Stable Transfection, Control, Incubation, Western Blot, Labeling

Journal: Molecular cell

Article Title: Members of an array of zinc finger proteins specify distinct Hox chromatin boundaries

doi: 10.1016/j.molcel.2024.08.007

Figure Lengend Snippet: Key Resources Table

Article Snippet: MEK1/2 inhibitor (PD0325901) , Stemgent , #04-0006-base.

Techniques: Virus, Recombinant, Magnetic Beads, SYBR Green Assay, Western Blot, Clone Assay, Plasmid Preparation, Software, Injection