Journal: Molecular Neurobiology

Article Title: NTF3 Is a Novel Target Gene of the Transcription Factor POU3F2 and Is Required for Neuronal Differentiation

doi: 10.1007/s12035-018-0995-y

Figure Lengend Snippet: Upregulation of the transcription factors POU3F2 and NTF3 during neuronal differentiation of NT2D1. a The protocol for neuronal induction of NT2D1 cells is schematized. b β3-tubulin staining for neuronal cells in NT2D1 cells untreated (non) and treated with neuronal induction medium at the indicated time points. c Quantification of β3-tubulin-positive cells. d Immunoblotting analysis for POU3F2, POU3F3, β3-tubulin, and NTF3 in NT2D1 cells untreated or treated with neuronal induction medium at the indicated time points. The values show the expression relative to that of untreated cells (to which a value of 1 was assigned). e Microarray analysis showed that neuronal induction for 6 h increased the expression of NTF3 and GADD45 in NT2D1 cells. f NTF3 mRNA expression after neuronal induction was analyzed by real-time PCR. The levels of mRNA were calculated as the relative expression compared with that of non-induced NT2D1 cells. GAPDH mRNA was used as a control. * p < 0.05; *** p < 0.001. g Phospho-TrkC (Tyr820) staining in treated and untreated NT2D1 cells. Values are presented as mean ± SEM of three independent experiments for c and f

Article Snippet: Human pluripotent embryonic carcinoma NTERA2 cl.D1 (NT2D1) cells (ATCC, CRL1973) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Staining, Western Blot, Expressing, Microarray, Real-time Polymerase Chain Reaction, Control

Journal: Molecular Neurobiology

Article Title: NTF3 Is a Novel Target Gene of the Transcription Factor POU3F2 and Is Required for Neuronal Differentiation

doi: 10.1007/s12035-018-0995-y

Figure Lengend Snippet: Identification of the POU3F2 binding site on the NTF3 promoter. a Transcription factor response elements predicted by the Transcription Element Search System for the nucleotide sequence of the NTF3 promoter region (− 1823 to + 243). The transcription start site is indicated as + 1. b Comparison of NTF3 promoter sequence conservation between different species. c Biotin-labeled oligonucleotides containing the intact or mutated POU3F2 binding site were hybridized with total lysates prepared from NT2D1 cells. The POU3F2-DNA complexes were precipitated by streptavidin agarose beads. POU3F2 was analyzed by Western blot analyses. The input of nuclear extracts was used as loading control. Three independent experiments were performed. d Chromatin was prepared from NT2D1 cells treated with induction medium for 0, 2, and 6 h. Cell lysates were mixed with antibodies against POU3F2 or IgG and then precipitated. The precipitates were analyzed by PCR for the presence of the NTF3 promoter sequence. The DNA purified from the sonicated chromatin was directly analyzed by PCR using the ChIP primer, which was used as an input control (Input). e The values of the ChIP DNA were normalized to that of the NT2D1 cells at 0 h (as a control). Values of fold-change over the control are presented as mean ± SEM of three independent experiments for d . * p < 0.05 compared with the control

Article Snippet: Human pluripotent embryonic carcinoma NTERA2 cl.D1 (NT2D1) cells (ATCC, CRL1973) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Binding Assay, Sequencing, Comparison, Labeling, Western Blot, Control, Purification, Sonication

Journal: Molecular Neurobiology

Article Title: NTF3 Is a Novel Target Gene of the Transcription Factor POU3F2 and Is Required for Neuronal Differentiation

doi: 10.1007/s12035-018-0995-y

Figure Lengend Snippet: Effects of POU3F2 on NTF3 promoter activity. a Schematic representation of NTF3-luciferase chimeric constructs. The negative numbers refer to the numbers of bases upstream of the transcription start (+ 1) site of the NTF3 gene. b NT2D1 cells were transiently transfected with the pGL3 basic vector or NTF3 promoter constructs of different lengths. The luciferase activity of each reporter was normalized to the Renilla luciferase activity and compared with that of cells transfected with the pGL3 basic vector (to which a value of 1 was assigned). *** p < 0.001. c NT2D1 cells were transfected with the pGL3 basic vector, pNTF3-1902, and pNTF3-1902 POU3F2 mut. Approximately 24 h later, cells were treated with neuronal induction medium. The transcriptional activity of each reporter was normalized to the Renilla luciferase activity and compared with that of cells transfected with the pGL3 basic vector (to which a value of 1 was assigned). *** p < 0.001. Values are presented as mean ± SEM of three independent experiments for b and c

Article Snippet: Human pluripotent embryonic carcinoma NTERA2 cl.D1 (NT2D1) cells (ATCC, CRL1973) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Activity Assay, Luciferase, Construct, Transfection, Plasmid Preparation

Journal: Molecular Neurobiology

Article Title: NTF3 Is a Novel Target Gene of the Transcription Factor POU3F2 and Is Required for Neuronal Differentiation

doi: 10.1007/s12035-018-0995-y

Figure Lengend Snippet: Effects of POU3F2 silencing on neuronal differentiation and NTF3 expression in NT2D1 cells. a POU3F2 expression in POU3F2-knockdown (shPOU3F2) and control (shLuc) NT2D1 cells was determined by Western blot analyses after neuronal induction for 6 h. GAPDH was used as a loading control. The values represent the relative expression compared with that of the non-induced shLuc cells (to which a value of 1 was assigned). b NTF3 mRNA expression of the cells described in a was analyzed by real-time PCR. mRNA levels were calculated relative to that of the non-induced shLuc cells. * p < 0.05; *** p < 0.001. c Neuronal morphology of shLuc and shPOU3F2 cells that were treated with neuronal induction medium for 24 h or left untreated (non). d Quantification of cell numbers of shLuc and shPOU3F2 described in c . All the percentages of the shLuc and shNTF3 cells were compared to that of the non-induction shLuc cells (to which a value of 100% was assigned). e β3-tubulin staining was performed on shLuc and shPOU3F2 cells, which were treated with neuronal induction medium for 0, 6, or 24 h or left untreated, after which neuronal cells were detected. Values represent the mean ± SEM of three independent experiments for b and d

Article Snippet: Human pluripotent embryonic carcinoma NTERA2 cl.D1 (NT2D1) cells (ATCC, CRL1973) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Expressing, Knockdown, Control, Western Blot, Real-time Polymerase Chain Reaction, Staining

Journal: Molecular Neurobiology

Article Title: NTF3 Is a Novel Target Gene of the Transcription Factor POU3F2 and Is Required for Neuronal Differentiation

doi: 10.1007/s12035-018-0995-y

Figure Lengend Snippet: Effects of NTF3 silencing and NTF3 recombinant protein treatment on the viability and neuronal differentiation of NT2D1 cells. a NTF3 mRNA levels in NTF3-knockdown (shNTF3) and control (shLuc) NT2D1 cells, which were treated with neuronal induction medium for 0, 24, or 48 h or left untreated (Non), were determined by real-time PCR. mRNA levels were calculated as the relative expression compared with the untreated shLuc cells. *** p < 0.001. b Phase contrast microscopy images of untreated shLuc and shNTF3 cells and those cells 24 h after neuronal induction with concomitant treatment of rNTF3 (5, 20 ng/ml) or vehicle. c Quantification of neuron number of shLuc and shNTF3 cells as described in b . All the percentages of neurons differentiated from shLuc and shNTF3 cells were compared to that of neurons differentiated from the vehicle-treated shLuc cells (to which a value of 100% was assigned). * p < 0.05; ** p < 0.01. d A suggested model of the POU3F2/NTF3 pathway that mediates the process of neuron differentiation. Values are presented as mean ± SEM of at least three independent experiments for a and c

Article Snippet: Human pluripotent embryonic carcinoma NTERA2 cl.D1 (NT2D1) cells (ATCC, CRL1973) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Recombinant, Knockdown, Control, Real-time Polymerase Chain Reaction, Expressing, Microscopy

Journal: bioRxiv

Article Title: Serotonin signaling by maternal neurons upon stress ensures progeny survival

doi: 10.1101/2020.01.20.913038

Figure Lengend Snippet: A, Experimental setup for assessing whether 5-HT is a conserved signal that activates HSF1 in mammalian cells. B, C, Time and dose-dependent change in Hspb1 and Hspb5 mRNA levels respectively, in control and 5-HT treated primary cortical neuronal cultures (n=4 experiments). D, Representative Western blot showing HSPA1A protein levels following 5-HT stimulation of NT2 cells (5µM was applied for 24 hrs to be able to assess protein accumulation; n=4 experiments). E, Quantitation of HSPA1A protein levels in control and 5-HT treated NT2 cells (n=3 experiments). Tubulin was used as internal control. F, Representative Western blot showing HSF1 protein levels in control and HSF1 siRNA treated NT2 cells to confirm siRNA knockdown of HSF1 (n=2 experiments). G, Temporal dynamics of HSPA1A mRNA levels in control and BIMU8 treated NT2 cells (n=5 experiments). H, HSPA1A mRNA levels in untreated and BIMU8 treated NT2 cells (10 µM for 10 minutes) transfected with control and HSF-1 siRNA (n=4 experiments). I, Hspa1 and Hspb1 mRNA levels in untreated and BIMU8 treated (10 µM for 10 minutes) primary cortical neuronal cultures (n=3 experiments). J, Quantitation of fluorescence intensity following immunostaining for HSF1 in the nuclei of untreated NT2 cells, NT2 cells treated with BIMU8 (10 µM for 10 minutes), and NT2 cells treated with BIMU8 and H89. Fluorescence intensity values (arbitrary units) were derived using ImageJ following background subtraction. (n=2 experiments; 25 cells). K, SUPT16H mRNA levels in control-siRNA and SUPT16H- siRNA treated NT2 cells to confirm siRNA knockdown of SUPT16H (n=5 experiments). Data in B, C, E, G-K show Mean ± Standard Error of the Mean. Values were normalized to that in either control untreated cells, or control-siRNA treated cells. *, p <0.05; **, p < 0.01 ***, p <0.001; (paired Students t-test).

Article Snippet: Control siRNA and siRNA targeting human HSF1 and SUPT16H (SPT16) were procured from Santa Cruz Biotechnology Inc, USA (catalog no. sc-37007, sc-35611 and sc-37875 respectively) and NT2 cells were transfected with Lipofectamine LTX Plus reagent according to manufacturer’s protocol.

Techniques: Control, Western Blot, Quantitation Assay, Knockdown, Transfection, Fluorescence, Immunostaining, Derivative Assay

Journal: bioRxiv

Article Title: Serotonin signaling by maternal neurons upon stress ensures progeny survival

doi: 10.1101/2020.01.20.913038

Figure Lengend Snippet: A, Time and dose-dependent change in Hspa1a mRNA levels in control and 5-HT treated primary cortical neuronal cultures (n=4 experiments). B, Dose-dependent change in HSPA1A mRNA levels in control NT2 cells and NT2 cells treated with 5-HT for 15 minutes (n=4 experiments). C, HSPA1A mRNA levels in NT2 cells treated with 5µM 5-HT for 15 minutes, transfected with control and HSF1 siRNA (n=4 experiments). D, HSPA1A mRNA levels in NT2 cells treated with two different doses of four 5-HT receptor agonists relative to control untreated cells (n=5 experiments). NT2 cells were treated for 10 minutes. E-F, Protein levels of S320 phospho-modified HSF1 in control NT2 cells and cells treated with 10µM BIMU8 for 10 minutes, in the presence or absence of the PKA inhibitor, H89 (n=4 experiments). E, Representative western blot using an antibody that recognizes HSF1 phosphorylated at S320. Tubulin served as the internal control. F, Quantitation of phospho-S320 levels (n=4 experiments). G, Representative micrographs showing projections of confocal images of HSF1 localization in control NT2 cells and cells treated with 10µM BIMU8 for 10 minutes, in the presence or absence of the H89 (n=2 experiments; 25 cells). Scale bar=10µm. H, HSPA1A mRNA levels relative to control NT2 cells upon treatment with 10µM BIMU8 for 10 minutes, in the presence or absence of H89 (n=5 experiments). I, HSPA1A mRNA levels in cells treated with 10µM BIMU8 for 10 minutes, transfected with control and SUPT16H siRNA. mRNA levels and protein levels are normalized to control RNAi-treated or unstimulated cells (n=5 experiments). Data in A-D, F, H, I show Mean ± Standard Error of the Mean. *, p <0.05; **, p < 0.01 ***, p <0.001; (Paired Student’s t-test). ns, non-significant.

Article Snippet: Control siRNA and siRNA targeting human HSF1 and SUPT16H (SPT16) were procured from Santa Cruz Biotechnology Inc, USA (catalog no. sc-37007, sc-35611 and sc-37875 respectively) and NT2 cells were transfected with Lipofectamine LTX Plus reagent according to manufacturer’s protocol.

Techniques: Control, Transfection, Modification, Western Blot, Quantitation Assay