Figure 1: HSF1 levels are decreased and P-Ser303/307 increased in HD. | Nature Communications

Figure 1: HSF1 levels are decreased and P-Ser303/307 increased in HD.

From: Abnormal degradation of the neuronal stress-protective transcription factor HSF1 in Huntington’s disease

Figure 1

(a) Diagram of the PC12-HttQ23 and Htt-Q74 proteins and experimental design. (b) Cells expressing either Htt-Q23 or Htt-Q74 were cultivated at 37 °C (c) in the presence of tetracycline (Dox) for 3 days to induce expression, exposed to heat shock 1 h at 42 °C, allowed to recover at 37 °C for 7 h (HS) and protein extracts immunoblotted with the indicated antibodies. HSF1 bands were quantitated using Quantity One image software (BioRad) and values normalized using GAPDH as loading control and referenced to control at 37 °C (c) in the absence of −Dox. (c) qRT-PCR analysis of the Hsp70 and Hsp25 genes in Htt-Q74-expressing cells as in B. HS (+Dox) group was compared with HS (−Dox) group. Error bars represent means±s.e.m., n=4. Unpaired t-test, ***P<0.001. (d) Analysis of Hsp70 promoter occupancy by HSF1. Error bars represent means±s.e.m., n=3. Unpaired t-test, **P<0.01. (e) Mouse-derived striatal STHdhQ7 and STHdhQ111 cells were cultured at 33 °C (c), heat shocked at 42 °C 1 h with recovery at 33 °C for 7 h and immunoblotted with the indicated antibodies. HSF1 was quantified using Quantity One image software and normalized using GAPDH as loading control and referenced to control at 37 °C in the non-pathogenic STHdhQ7. (f) Striatal samples from Wild type (WT) C57BL/6 or KIQ175 mice at 2, 6 and 12 months analysed by immunoblotting (n=4). (g) Dorsal striatal sections from WT and KIQ175 mice (n=3) at 6 months assayed by immunohistochemistry (IHC) for mHtt aggregation (1C2), HSF1 and HSF1-S303 phosphorylation using DAPI staining as control. Scale bar: 10 μm. (h) Gastrocnemius muscle extracts from WT and KI175 mice (n=4) of the indicated age, immunoblotted for HSF1, Hsp70 and GAPDH. (i) Protein extracts from HD patient striatum (Supplementary Table 1) and controls immunoblotted with indicated antibodies. (j) HSF1 phosphoproteomic analysis under non-pathogenic (−Dox) and pathogenic (+Dox) conditions in hsf1−/− MEF inducible cell line expressing GFP-Htt-Q74 expressing HSF1. See Supplementary Fig. 8 for uncropped immunoblots. HSF1 represented by the Regulatory domain, DBD; DNA binding domain; LZ1-3 and LZ4; leucine zipper domains.

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