Journal: Cell Reports

Article Title: The transcription factor ZEB1 regulates stem cell self-renewal and cell fate in the adult hippocampus

doi: 10.1016/j.celrep.2021.109588

Figure Lengend Snippet: ZEB1 directly regulates expression of ETV5 (A) Workflow for narrowing down the list of candidates relevant for asymmetric division. (B) Predicted ZEB1 binding sites with a p value of 10 −3 (based on the JASPAR database; Fornes et al., 2020 ) in the Etv5 promoter region. (C) ChIP of the Etv5 promoter after pulldown with ZEB1 from hippocampal neurosphere cultures. (D) Western blot of ETV5 and ZEB1 in hippocampal Zeb1 −/− and control neurospheres. (E) Immunofluorescence staining for ETV5 in RGL cells of control and Zeb1 −/− mice. (F) Quantification of ETV5 expression in control RGL cells. (G) Quantification of ETV5 + GFAP + tdTOM + cells with RGL morphology in the SGZ at 1 day post-induction. (H) Percentage of ETV5 + GFAP + tdTOM + RGL cells out of total GFAP + tdTOM + RGL cells at 1 day post-induction. (I) Western blot of ETV5 in hippocampal Zeb1 −/− neurospheres transduced with a lentiviral ETV5 expression vector and control Zeb1 −/− cultures. (J) Ratio of asymmetric to symmetric divisions quantified from time-lapse imaging of primary adult hippocampal cells. (K) Quantification of GFAP + and DCX + cells after live-cell imaging. Dots represent individual mice (minimum of two sections analyzed per animal); numerical data are shown as mean ± SEM. Scale bars: 10 μm. GBM, glioblastoma; HC, hippocampus. See also Figure S5 and .

Article Snippet: Lentiviral plasmid for ETV5 expression (pLX_TRC311 ETV5) , , Addgene plasmid # 74984; http://addgene.org/74984/ ; RRID:Addgene_74984.

Techniques: Expressing, Binding Assay, Western Blot, Control, Immunofluorescence, Staining, Transduction, Plasmid Preparation, Imaging, Live Cell Imaging

Journal: Cell Reports

Article Title: The transcription factor ZEB1 regulates stem cell self-renewal and cell fate in the adult hippocampus

doi: 10.1016/j.celrep.2021.109588

Figure Lengend Snippet:

Article Snippet: Lentiviral plasmid for ETV5 expression (pLX_TRC311 ETV5) , , Addgene plasmid # 74984; http://addgene.org/74984/ ; RRID:Addgene_74984.

Techniques: Virus, Plasmid Preparation, Expressing, Recombinant, Software, Microscopy

Journal: Cell

Article Title: Loss of Ataxin-1 Potentiates Alzheimer's Pathogenesis by Elevating Cerebral BACE1 Transcription

doi: 10.1016/j.cell.2019.07.043

Figure Lengend Snippet: (A) Actinomycin D or vehicle (DMSO) was treated for 48 hrs in acute brain slice cultures. Results are from two independent brain slice cultures (2 mice each for WT and Ataxin-1 KO). n (brain slices) = 3–4. *p < 0.05, **p < 0.01, ***p < 0.001, t-test. (B) Left: extracellular field potential records of multiple unit activity in CA1 areas of brain slices 48 hrs after incubation. Right: histograms representing spike amplitude distribution (bin size 10 mV) and inter-spike interval (bin size 2 ms). (C) Representative real-time qPCR amplification graphs of steady-state (total) and nascent Bace1 mRNA. Brain slices were incubated for 16 hrs in ACSF containing 5-ethynyl uridine. n (brain slices) = 4. (D) Locations of predicted CIC and PEA3 (ETV1/4/5) binding sites in Etv1/4/5 and Bace1 promoter regions, respectively. The CIC binding site in Etv1 promoter is relative distal (−1486) than those in Etv4/5. (E) CIC expression in medial cortex (left) and hippocampal CA1 (right). Bar = 50 μm. (F) mRNA levels of Etv1, Etv4, and Etv5 in the cortex and cerebellum. Gapdh was used as an internal control for RT-qPCR.All levels were presented as relative to Etv1-WT (100). Compared to cortex, the relative levels of Etv4 and Etv5 versus Etv1 mRNA are very low in cerebellum. n= 6 (cortex), 4 (cerebellum). (G) Western blot analysis for cortical levels of Ataxin-1, CIC, ETV4, ETV5, BACE1, sAPPβ, and sAPPα. (H) Cerebellar levels of ETV4 and ETV5. Vertical lines indicate that different parts within same blot were placed together for better comparison. (I) Densitometric quantification. n = 5, except ETV4 and ETV5 (n = 7). (J) Luciferase activity in HEK293T cells measured 24 hr after transfection with pGL3 plasmids harboring BACE1 promoter and vectors expressing either ETV4 or ETV5. Results from 3 independent experiments of sextuplicate transfection were normalized and combined. n = 18. See also Figure S2.

Article Snippet: One day after seeding, the cells were transfected with 100 ng pGL3-BACE1 promoter, 50 ng pRL-TK (for renilla luciferase expression, Promega), and either 200 ng of pLX_TRC311 (Addgene #113668), pLX_TRC311 ETV4-S (Addgene #74983), pLX_TRC311 ETV4-L (Addgene #74982), or pLX_TRC311 ETV5 (Addgene #74984) using jetPrime transfection reagent (Polyplus transfection) according to manufacturer’s instructions.

Techniques: Slice Preparation, Activity Assay, Incubation, Amplification, Binding Assay, Expressing, Quantitative RT-PCR, Western Blot, Luciferase, Transfection

Journal: Cell

Article Title: Loss of Ataxin-1 Potentiates Alzheimer's Pathogenesis by Elevating Cerebral BACE1 Transcription

doi: 10.1016/j.cell.2019.07.043

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: One day after seeding, the cells were transfected with 100 ng pGL3-BACE1 promoter, 50 ng pRL-TK (for renilla luciferase expression, Promega), and either 200 ng of pLX_TRC311 (Addgene #113668), pLX_TRC311 ETV4-S (Addgene #74983), pLX_TRC311 ETV4-L (Addgene #74982), or pLX_TRC311 ETV5 (Addgene #74984) using jetPrime transfection reagent (Polyplus transfection) according to manufacturer’s instructions.

Techniques: Recombinant, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Luciferase, Reporter Assay, Staining, Expressing, Knock-In, SYBR Green Assay, Clone Assay, Mutagenesis, Software, Microscopy, Real-time Polymerase Chain Reaction, Transfection

Journal: Cell

Article Title: Loss of Ataxin-1 Potentiates Alzheimer's Pathogenesis by Elevating Cerebral BACE1 Transcription

doi: 10.1016/j.cell.2019.07.043

Figure Lengend Snippet: (A) Actinomycin D or vehicle (DMSO) was treated for 48 hrs in acute brain slice cultures. Results are from two independent brain slice cultures (2 mice each for WT and Ataxin-1 KO). n (brain slices) = 3–4. *p < 0.05, **p < 0.01, ***p < 0.001, t-test. (B) Left: extracellular field potential records of multiple unit activity in CA1 areas of brain slices 48 hrs after incubation. Right: histograms representing spike amplitude distribution (bin size 10 mV) and inter-spike interval (bin size 2 ms). (C) Representative real-time qPCR amplification graphs of steady-state (total) and nascent Bace1 mRNA. Brain slices were incubated for 16 hrs in ACSF containing 5-ethynyl uridine. n (brain slices) = 4. (D) Locations of predicted CIC and PEA3 (ETV1/4/5) binding sites in Etv1/4/5 and Bace1 promoter regions, respectively. The CIC binding site in Etv1 promoter is relative distal (−1486) than those in Etv4/5. (E) CIC expression in medial cortex (left) and hippocampal CA1 (right). Bar = 50 μm. (F) mRNA levels of Etv1, Etv4, and Etv5 in the cortex and cerebellum. Gapdh was used as an internal control for RT-qPCR.All levels were presented as relative to Etv1-WT (100). Compared to cortex, the relative levels of Etv4 and Etv5 versus Etv1 mRNA are very low in cerebellum. n= 6 (cortex), 4 (cerebellum). (G) Western blot analysis for cortical levels of Ataxin-1, CIC, ETV4, ETV5, BACE1, sAPPβ, and sAPPα. (H) Cerebellar levels of ETV4 and ETV5. Vertical lines indicate that different parts within same blot were placed together for better comparison. (I) Densitometric quantification. n = 5, except ETV4 and ETV5 (n = 7). (J) Luciferase activity in HEK293T cells measured 24 hr after transfection with pGL3 plasmids harboring BACE1 promoter and vectors expressing either ETV4 or ETV5. Results from 3 independent experiments of sextuplicate transfection were normalized and combined. n = 18. See also Figure S2.

Article Snippet: One day after seeding, the cells were transfected with 100 ng pGL3-BACE1 promoter, 50 ng pRL-TK (for renilla luciferase expression, Promega), and either 200 ng of pLX_TRC311 (Addgene #113668), pLX_TRC311 ETV4-S (Addgene #74983), pLX_TRC311 ETV4-L (Addgene #74982), or pLX_TRC311 ETV5 (Addgene #74984) using jetPrime transfection reagent (Polyplus transfection) according to manufacturer’s instructions.

Techniques: Slice Preparation, Activity Assay, Incubation, Amplification, Binding Assay, Expressing, Quantitative RT-PCR, Western Blot, Luciferase, Transfection

Journal: Cell

Article Title: Loss of Ataxin-1 Potentiates Alzheimer's Pathogenesis by Elevating Cerebral BACE1 Transcription

doi: 10.1016/j.cell.2019.07.043

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: One day after seeding, the cells were transfected with 100 ng pGL3-BACE1 promoter, 50 ng pRL-TK (for renilla luciferase expression, Promega), and either 200 ng of pLX_TRC311 (Addgene #113668), pLX_TRC311 ETV4-S (Addgene #74983), pLX_TRC311 ETV4-L (Addgene #74982), or pLX_TRC311 ETV5 (Addgene #74984) using jetPrime transfection reagent (Polyplus transfection) according to manufacturer’s instructions.

Techniques: Recombinant, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Luciferase, Reporter Assay, Staining, Expressing, Knock-In, SYBR Green Assay, Clone Assay, Mutagenesis, Software, Microscopy, Real-time Polymerase Chain Reaction, Transfection