Figure 2 | Scientific Reports

Figure 2

From: Transcriptional induction of the heat shock protein B8 mediates the clearance of misfolded proteins responsible for motor neuron diseases

Figure 2

Development of the cellular system for the screening of the HSPB8 promoter modulators (A) Validation of the GFP-hPromB8LUC plasmid.

NSC34 cells were transiently transfected with GFP-hPromB8LUC and pRL-TK plasmids. 24 hrs after transfection, cells were treated with DMSO or 10 μM MG132 for the last 16 hrs prior to luminescence analysis. The histogram represents the Luminescence Counts Per Second (LCPS) of the inducible firefly luciferase normalized by the LCPS of renilla luciferase of six independent replicates (**p < 0.01 vs DMSO). (B) Clones selection of NSC34-GFP-hPromB8LUC (#Ns) stably transfected cells. Different #Ns clones were analysed for their luminescence levels, 48 hrs after plating. NSC34 untransfected cells were used as negative control. The histogram represents the firefly luciferase LCPS mean normalized by the total protein level mean of six independent replicates for each clone (**p < 0.01 vs NSC34 untransfected cells). (C) Optimization of cell number and signal to background ratio for HTS assay. Raw data of relative luminescence units detected by NSC34 clones after 24 hrs seeding at the indicated cell number. SD refers to two independent experiments. (D) Validation of the #N4 selected clone. NSC34-GFP-hPromB8LUC #N4 cells were analysed for their ability to respond to MG132 treatment. 24 hrs after plating, cells were treated with DMSO or 10 μM MG132 for the last 16 hrs prior to luminescence analysis. The histogram represents the firefly inducible luciferase LCPS mean normalized by the total protein level mean of six independent replicates (**p < 0.01 vs DMSO).

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