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Fig. 4 | Molecular Brain

Fig. 4

From: Glucocorticoid receptor represses brain-derived neurotrophic factor expression in neuron-like cells

Fig. 4

GR-dependent inhibitory effects on BDNF promoter 4 activity relies on a short sequence upstream of exon IV. a, Map of transfected constructs, LP6, SP6, LP4 and SP4 regions. These sequences were subcloned into PGL4 luciferase plasmid, setting the first base of exon VI at +1: a long (LP6, 1.8 kb, -1563, +227, containing exon IV, V and part of VI) and three short regions (SP6, 837 bp, -610, +227, partly encompassing exon VI; LP4, 543 bp, -1563, -1021; SP4, 275 bp, -1315, -1041; LP4 and SP4 are upstream of exon IV and include its transcription start site). b, DEX dose responses (Veh, Ethanol for 24 h) of SP6 transfected in N2A cells, co-transfected with GR expression vector and Renilla plasmid for normalization. Results are expressed as fold change of the luciferase/Renilla value of SP6 transfected alone. No significance was observed whatsoever (n = 16), showing SP6-driven activities were not modified by DEX. c, Same as in (b) with LP6 construct, Veh condition is set at 1; n = 16, Mean ± SEM; # p < 0.05, LP6 vs Veh; **P < 0.01, ***P < 0.005, DEX vs Veh; Mann Whitney U-tests. Luciferase activities driven by LP6 were already decreased by DEX at a low concentration. d, Transfection of LP6 in N2A cells, 24 h treatment with Veh, DEX (10-7M), RU (10-6M) and both; n = 16, Mean ± SEM; $ p < 0.05, $$ p < 0.01, vs DEX; Mann Whitney U-tests. The DEX repression is inhibited by RU. e, DEX dose responses of LP4 transfected in N2A cells. Results are expressed as fold change of the luciferase/protein value of LP4 transfected alone. Veh condition is set at 1; n = 8; ## P < 0.01, LP4 vs Veh;* P < 0.05, ** P < 0.01, DEX vs Veh; Mann Whitney U-tests. DEX reduced the LP4 activities in a dose-response manner. f, Transfection of LP4 construct plasmid in N2A cells, 24 h treatment with Veh, DEX (10-7M), RU (10-6M) or both; n = 16, Mean ± SEM; *P < 0.05 vs Veh; $ p < 0.05, vs DEX; Mann Whitney U-tests. The DEX effect was not reversed by RU. g, Same as in (e) with SP4 construct. DEX vs Veh; * P < 0.05 and ** P < 0.01. DEX repressed SP4 activity. h, SP4 fragment is repressed by GR. Same as in (f) with SP4 construct; ** P < 0.01 vs Veh; $$ P < 0.01 vs DEX

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