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

Journal: Scientific Reports

Article Title: CRISPR/Cas9-mediated deletion of a GA-repeat in human GPM6B leads to disruption of neural cell differentiation from NT2 cells

doi: 10.1038/s41598-024-52675-3

Figure Lengend Snippet: In-silico analysis of GPM6B expression. ( a ) Across several primates, the expression of GPM6B was at its highest level in the human brain. ( b ) Our analysis of the RNA-seq datasets retrieved from GEO showed a positive correlation between GPM6B and neural cell differentiation markers, such as GFAP, TUBB3, and MAP2, after differentiation of NT2 cells into neural cells (21 days under RA treatment). HUM, human; CHP, chimpanzee; OWM, Old-World monkeys; NWM, New-World monkeys; MLM, mouse lemur.

Article Snippet: NTERA-2 (NT2) cell line (Cat. #ACC-527, RRID:CVCL_0034; was purchased from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH), resembling characteristics of human neuronal progenitor cell, cultured in high glucose DMEM supplemented with 1% penicillin-streptomycin, and 10% FBS under optimal growth condition (at 37 °C, 5% CO 2 , 80% humidity).

Techniques: In Silico, Expressing, RNA Sequencing, Cell Differentiation

Journal: Scientific Reports

Article Title: CRISPR/Cas9-mediated deletion of a GA-repeat in human GPM6B leads to disruption of neural cell differentiation from NT2 cells

doi: 10.1038/s41598-024-52675-3

Figure Lengend Snippet: Measuring the expression level of the edited GPM6B gene at the RNA and protein levels in NT2 cells. ( a ) The expression level of GPM6B was evaluated in the untreated and edited NT2 pool cells, using qRT-PCR. The expression level of GPM6B decreased significantly in the edited pool cells ( p < 0.05). ( b ) The expression of GPM6B was assessed in the isolated single clones, using qRT-PCR. The expression of GPM6B was significantly decreased in the C1 cells, compared to the untreated and the SC3 cells. ( c ) Western blotting assay confirmed that the expression level of GPM6B was decreased in the C1 cells more efficiently, compared to the untreated and SC3 cells. The original blot images are presented in . ( d ) the predicted TF binding sites at GA-repeat site and its flanking sequence, is presented based on JASPAR CORE 2022 and ChIP-seq data from ENCODE. The GA-repeat site is highlighted with light blue.

Article Snippet: NTERA-2 (NT2) cell line (Cat. #ACC-527, RRID:CVCL_0034; was purchased from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH), resembling characteristics of human neuronal progenitor cell, cultured in high glucose DMEM supplemented with 1% penicillin-streptomycin, and 10% FBS under optimal growth condition (at 37 °C, 5% CO 2 , 80% humidity).

Techniques: Expressing, Quantitative RT-PCR, Isolation, Clone Assay, Western Blot, Binding Assay, Sequencing, ChIP-sequencing

Journal: Scientific Reports

Article Title: CRISPR/Cas9-mediated deletion of a GA-repeat in human GPM6B leads to disruption of neural cell differentiation from NT2 cells

doi: 10.1038/s41598-024-52675-3

Figure Lengend Snippet: Decreased level of the GPM6B due to the GA-repeat deletion reduced differentiation of NT2 cells. ( a ) Differentiation of NT2, SC3, and C1 cells was monitored under RA treatment on Days 0, 4, 8, 12, 16, and 21. DMSO was used to dissolve RA, and was added to NT2 cells to evaluate its background effect of cell differentiation. On different days, the morphological changes and cell communication among differentiated cells were observed in NT2 and SC3 cells, whereas the morphology of C1 cells mainly remained undifferentiated. ( b ) The expression of TUBB3 and MAP2 was measured at the RNA level, using qRT-PCR. The expression of these genes was significantly decreased in the C1 compared to the SC3 cells. ( c ) The expression of neural markers was measured on Day 0 and Day 21 of RA-treatment, using western blotting (the original blot images are presented in ). The expression of NES as an indicator of neural progenitor cells increased, while the expression of GFAP, TUBB3, and MAP2, as differentiated neural markers, were decreased in the C1 compared to the SC3 cells.

Article Snippet: NTERA-2 (NT2) cell line (Cat. #ACC-527, RRID:CVCL_0034; was purchased from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH), resembling characteristics of human neuronal progenitor cell, cultured in high glucose DMEM supplemented with 1% penicillin-streptomycin, and 10% FBS under optimal growth condition (at 37 °C, 5% CO 2 , 80% humidity).

Techniques: Cell Differentiation, Expressing, Quantitative RT-PCR, Western Blot

Journal: Scientific Reports

Article Title: CRISPR/Cas9-mediated deletion of a GA-repeat in human GPM6B leads to disruption of neural cell differentiation from NT2 cells

doi: 10.1038/s41598-024-52675-3

Figure Lengend Snippet: Disrupted differentiation of NT2 cells to astrocytic and neural cells as a result of GA-repeat deletion and GPM6B downregulation. Deletion of the GA-repeat in the regulatory region of GPM6B decreased the expression of this gene. Consequently, the number of differentiated cells expressing GFAP, TUBB3, and MAP2 decreased significantly in the C1 compared to the SC3 cells.

Article Snippet: NTERA-2 (NT2) cell line (Cat. #ACC-527, RRID:CVCL_0034; was purchased from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH), resembling characteristics of human neuronal progenitor cell, cultured in high glucose DMEM supplemented with 1% penicillin-streptomycin, and 10% FBS under optimal growth condition (at 37 °C, 5% CO 2 , 80% humidity).

Techniques: Expressing