Journal: Molecular and cellular endocrinology

Article Title: SDHB and SDHD silenced pheochromocytoma spheroids respond differently to tumour microenvironment and their aggressiveness is inhibited by impairing stroma metabolism.

doi: 10.1016/j.mce.2022.111594

Figure Lengend Snippet: Fig. 1. Characterization of stable SDHB and SDHD silenced cell lines. (A) Representative blots of the expression of mitochondrial SDHA, B and D subunits. (B) Densitometric analysis of western blot bands, performed by Bio-Rad imaging and analysis software (Quantity One), showed significant differences in the SDHB and SDHD subunit expression levels in SDHB (light grey) and in SDHD silenced cells (dark grey) respectively, compared to Wt (black). Bars are the means of three independent preparations ± SD, ***p < 0.001. (C) Representative traces of SDH enzymatic activity measured in cell homogenates. The silenced SDHB and SDHD cells (dotted and continuous lines, respectively) showed a similar decrease of the SDH activity, significantly different compared with Wt (dashed line). (D) Histogram represents the SDH activity expressed as the percentages. SDHB and SDHD silenced cells (light and dark grey, respectively) showed a significantly decreased of SDH activity compared to Wt (black). Bars are the means of three independent experiments (each of them conducted in duplicate samples) ± SD, ***p < 0.01. (E) The bar graph represents the means of intracellular succinate/fumarate ratio ± SD, measured by GC/MS, in three independent experiments with two replicates. SDHB silenced cells (light grey) showed a significant increase of the metabolites ratio compared with both Wt (black) and to SDHD silenced cells (dark grey), *p < 0.05, **p < 0.01. (F) Representative immunoblot of HIF1α expression in Wt, SDHB and SDHD silenced cells. (G) Optical density analysis of western blot bands. Actin im munoblots was used as loading control. For all the analyses One-way ANOVA post-test Bonferroni was used.

Article Snippet: Bound antibodies were detected using ECL reagents (Immobilon) and analysed with a Bio-Rad ChemiDoc Imaging System (Quantity One) for dedicated chemiluminescent image acquisition.

Techniques: Expressing, Western Blot, Imaging, Software, Activity Assay, Gas Chromatography-Mass Spectrometry, Control

Journal: Nature Communications

Article Title: Abnormal degradation of the neuronal stress-protective transcription factor HSF1 in Huntington's disease

doi: 10.1038/ncomms14405

Figure Lengend Snippet: ( 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 STHdh Q7 and STHdh Q111 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 STHdh Q7 . ( 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 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 for uncropped immunoblots. HSF1 represented by the Regulatory domain, DBD; DNA binding domain; LZ1-3 and LZ4; leucine zipper domains.

Article Snippet: HttQ74 cells were transfected with HA-Ub vector (Addgene #18712) by using Lipofectamine LTX Reagent (Invitrogen) following protocol instructions (Invitrogen protocols 25-0946 W). hsf1 −/− MEF-GFP-Htt-Q74 cells were transfected with pcDNA WT HSF1 or the HSF1 S303A mutant plasmid using Lipofectamine LTX reagent. hsf1 −/− MEFs were electroporated with FBXW7-FLAG tag and WT HSF1 or S303A plasmids using the SE Cell Line Nucleofector.

Techniques: Expressing, Software, Quantitative RT-PCR, Derivative Assay, Cell Culture, Western Blot, Immunohistochemistry, Staining, Binding Assay

Journal: Nature Communications

Article Title: Abnormal degradation of the neuronal stress-protective transcription factor HSF1 in Huntington's disease

doi: 10.1038/ncomms14405

Figure Lengend Snippet: ( a , b ) PC12 cells expressing Htt-Q74 for 1, 2 or 3 days followed by Heat Shock (HS) and recovery as indicated. Control and HS in the absence of Dox correspond to cells incubated during 3 days at 37 °C. ( c ) Htt-Q74 cells Dox-induced or not for 3 days and exposed to 2 μM 17-AAG and/or MG132 (5 μM) for 6 h and extracts immunoblotted with the indicated antibodies. ( d ) Diagram of the effects of 17-AAG and MG132 treatment and Htt-Q74 expression in HSF1. ( e ) Htt-Q74 cells transfected with human influenza haemagglutinin-ubiquitin (HA-Ub) plasmid Dox induced or not and treated with 5 μM MG132 treatment for 6 h. Whole-cell extract and HA immunoprecipitated (IP:HA) and HSF1 immunoprecipitated samples (IP:HSF1) were immunoblotted as indicated. ( f ) Transcript levels for indicated E3 ligases evaluated by qRT-PCR from striatum of WT and KIQ175 mice at 6 months. Error bars represent±s.e.m., ( n =3). Unpaired t -test, * P <0.05. ( g ) Human striatum samples from HD patients and controls and ( h ) Mouse striatum from 12 months old WT and KIQ175 mice were immunoblotted for HSF1 and Fbxw7. ( i ) Fbxw7 siRNA in STHdh Q7 and STHdh Q111 cells using scrambled RNA (Scr) as control. 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 STHdh Q7 . ( j ) hsf1 −/− MEFs transfected with WT HSF1 or S303A mutant and Fbxw7-FLAG; samples immunoprecipitated with anti-FLAG and HSF1 detected. ( k ) hsf1 −/− MEFs expressing Dox-inducible Htt-Q74-GFP transfected with WT HSF1 or HSF1-S303A HS and 1 h recovery at 37 °C for 7 h. HSF1 was quantified using Quantity One image software and normalized using GAPDH as loading control and referenced to control (−Dox) expressing WT HSF1. See for uncropped immunoblots.

Article Snippet: HttQ74 cells were transfected with HA-Ub vector (Addgene #18712) by using Lipofectamine LTX Reagent (Invitrogen) following protocol instructions (Invitrogen protocols 25-0946 W). hsf1 −/− MEF-GFP-Htt-Q74 cells were transfected with pcDNA WT HSF1 or the HSF1 S303A mutant plasmid using Lipofectamine LTX reagent. hsf1 −/− MEFs were electroporated with FBXW7-FLAG tag and WT HSF1 or S303A plasmids using the SE Cell Line Nucleofector.

Techniques: Expressing, Incubation, Transfection, Plasmid Preparation, Immunoprecipitation, Quantitative RT-PCR, Software, Mutagenesis, Western Blot

Journal: Nature Communications

Article Title: Abnormal degradation of the neuronal stress-protective transcription factor HSF1 in Huntington's disease

doi: 10.1038/ncomms14405

Figure Lengend Snippet: ( a ) Mammalian CK2 holoenzyme subunit composition and function. ( b ) Htt-Q74 cells treated with CK2 kinase inhibitors TID43 or ( c ) Emodin 24 h before Htt-Q74 induction with Dox and heat shocked at 42 °C for 1 h followed by recovery at 37 °C for 7 h and extracts analysed by immunoblotting for Hsp70 and GAPDH. ( d ) Htt-Q74 cells treated with 5 μM TID43 and immunoblotted for HSF1 and P-HSF1-S303. ( e ) Fluorescent images for GFP-Htt-Q74 analysed microscopically in cells treated with DMSO or 1 μM TID43 as described in B. Scale bar: 200 μm. ( f ) Quantification of cells containing GFP-Htt-Q74 aggregates from E expressed as percentage of total number of cells evaluated. Error bars represent±s.e.m., ( n =500 cells). Unpaired t -test ** P <0.05. ( g ) hsf1 −/− MEFs expressing Dox-inducible Htt-Q74-GFP transfected with pcDNA or WT HSF1 and incubated with 1 μM TID43 24 h before Htt-Q74-GFP induction followed by heat shock at 42 °C during 1 and 7 h recovery at 37 °C. Cell viability expressed as % of viable cells under control conditions at 37 °C. Error bars represent±s.e.m., ( n =3). Unpaired t -test n.s., no significant, * P <0.05, ** P <0.01). ( h ) Htt-Q74 cells were transfected with siRNA against CK2β regulatory subunit or ( i ) CK2α and/or CK2α′ catalytic subunits using scrambled siRNA (Scr) as control 24 h before Htt-Q74 induction during 2 days followed by heat shock at 42 °C 1 h and recovery at 37 °C, 7 h. HSF1 was quantitated as in (F2H). All immunoblots shown for each panel contain the samples from the same membrane and were cropped to show only relevant data. See for uncropped immunoblots.

Article Snippet: HttQ74 cells were transfected with HA-Ub vector (Addgene #18712) by using Lipofectamine LTX Reagent (Invitrogen) following protocol instructions (Invitrogen protocols 25-0946 W). hsf1 −/− MEF-GFP-Htt-Q74 cells were transfected with pcDNA WT HSF1 or the HSF1 S303A mutant plasmid using Lipofectamine LTX reagent. hsf1 −/− MEFs were electroporated with FBXW7-FLAG tag and WT HSF1 or S303A plasmids using the SE Cell Line Nucleofector.

Techniques: Western Blot, Expressing, Transfection, Incubation

Journal: Nature Communications

Article Title: Abnormal degradation of the neuronal stress-protective transcription factor HSF1 in Huntington's disease

doi: 10.1038/ncomms14405

Figure Lengend Snippet: ( a ) CK2α, CK2α′ and CK2β protein levels in Htt-Q74 expressing cells under control ( c ) or heat shock conditions at 42 °C for 1 h (HS). CK2 subunit abundance was quantified using Quantity One image software normalized using GAPDH as loading control. CK2α′ ratio is shown and referenced to control (−Dox) cells. ( b ) CK2α, CK2α′ and CK2β striatal mRNA levels from WT and KIQ175 mice at 6 months of age. The value given for the amount of mRNA in the control group (WT) was set as 1. Error bars represent mean±s.e.m., ( n =4 animals). Values for the KIQ175 group were compared to the WT group. Statistical significance was measured by two-tailed unpaired t -test * P <0.05. ( c ) Protein levels for CK2α, CK2α′ and CK2β in the striatum and ( d ) gastrocnemius muscle of WT and KIQ175 mice at 6 months of age ( n =4). ( e ) Coronal section of the striatum of WT and KIQ175 at 6 months of age, showing co-localization of CK2α′ (red) with Ctip2 (green) and Fox1p (magenta) labelled MSNs in merged image. Scale bar: 10 μm. ( f ) CK2α, CK2α′ and CK2β qRT-PCR analysis and ( g ) protein levels in the striatum of HD patients and sex-age matched controls from 3 biospecimen banks . The value given for the amount of mRNA in the control group (C) was set as 1 for each gene. Error bars represent mean±s.e.m., ( n =7). One-tailed unpaired t -test * P <0.05, ** P <0.05, NS, no significant. Values for the Huntington's disease (HD) group were compared to the control (C) group. CK2α′ bands from immunoblots were quantified using Quantity One image software (BioRad) and the protein values were normalized using GAPDH as loading control and referenced to the corresponding age-sex-matched control patient. See for uncropped immunoblots.

Article Snippet: HttQ74 cells were transfected with HA-Ub vector (Addgene #18712) by using Lipofectamine LTX Reagent (Invitrogen) following protocol instructions (Invitrogen protocols 25-0946 W). hsf1 −/− MEF-GFP-Htt-Q74 cells were transfected with pcDNA WT HSF1 or the HSF1 S303A mutant plasmid using Lipofectamine LTX reagent. hsf1 −/− MEFs were electroporated with FBXW7-FLAG tag and WT HSF1 or S303A plasmids using the SE Cell Line Nucleofector.

Techniques: Expressing, Software, Two Tailed Test, Quantitative RT-PCR, One-tailed Test, Western Blot