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