Journal: EMBO Reports

Article Title: HSC70 coordinates COP9 signalosome and SCF ubiquitin ligase activity to enable a prompt stress response

doi: 10.1038/s44319-025-00376-x

Figure Lengend Snippet: ( A ) In vitro deneddylation assay. CSN deneddylation activity in the presence or absence of 500 nM HSC70. Neddylated or non-neddylated CUL1 were detected by immunoblotting with anti-CUL1 antibody. The differences in the deneddylation activity of CSN were illustrated by estimating the percentage of non-neddylated CUL1 to total CUL1 at each time point based on protein band intensities. The bottom graphs show the mean ± s.e.m. * P < 0.05 ( n = 3 independent experiments, respectively; Welch’s t test). A significant difference was observed at 20 min ( P = 0.014) and 40 min ( P = 0.044). ( B ) In vitro deneddylation assay. CSN deneddylation activity in the presence of 500 nM HSC70 with or without 5 µM NR peptide. The bottom graphs show the mean ± s.e.m. ** P < 0.01 ( n = 3 independent experiments, respectively; Welch’s t test). A significant difference was observed at 20 min ( P = 0.005) and 40 min ( P = 0.0023). ( C ) In vitro pulldown assay. A mixture containing Strep-tagged CSN and GST-tagged HSC70 WT or E175S mutant was subjected to immunoprecipitation with Streptactin Sepharose. ( D ) In vitro deneddylation assay. CSN deneddylation activity in the presence or absence of 500 nM HSC70 E175S. The bottom graphs show the mean ± s.e.m. * P < 0.05 ( n = 3 independent experiments, respectively; Welch’s t test). A significant difference was observed at 40 min ( P = 0.036). ( E ) Immunoblotting analysis of the established HSC70 knockdown (shHSC70) cell line. GAPDH protein was used as an internal control, and bar graphs show the relative expression level of HSC70 in shHSC70 cells to those in shCtrl cells ( n = 6 independent expermeriments), and the ratio of neddylated CUL1 to total CUL1 in shCtrl and shHSC70 cells ( n = 9 independent experiments). The box represents the interquartile range (IQR), with the bottom and top edges indicating the 25th and 75th percentiles, respectively. The horizontal line within the box denotes the median (50th percentile). The whiskers extend to the minimum and maximum values from the box. Data points are shown individually. ( F ) Immunoblotting analysis of shCtrl and shHSC70 cells with or without transient FLAG-HSC70 expression. .

Article Snippet: Lentiviral particle production involved transfecting plasmids encoding shCtrl or shHSC70 in the pLKO.1 puromycin resistant vector or pCDH-CMV-PA-HA-CSN3-EF1 purovector into HEK293T cells together with pRSV-Rev (Addgene no. 12253), pMD 2.G (Addgene no. 12259), and pMDL g/p RRE plasmids (Addgene no. 12251) using Lipofectamine 2000.

Techniques: In Vitro, Activity Assay, Western Blot, Mutagenesis, Immunoprecipitation, Knockdown, Control, Expressing

Journal: EMBO Reports

Article Title: HSC70 coordinates COP9 signalosome and SCF ubiquitin ligase activity to enable a prompt stress response

doi: 10.1038/s44319-025-00376-x

Figure Lengend Snippet: ( A ) CHX chase assay of endogenous MCL1 with pharmacological inhibition with YM-01 or VER155008. Each protein level was densitometrically quantified (normalized to 0 min) and shown in the graph below. The bottom graph shows the mean ± s.e.m. * P < 0.05, *** P < 0.001 ( n = 3 independent experiments, respectively; Tukey’s HSD test.). Compared with DMSO treatment, a significant difference was observed at 60 min ( P = 0.014), 120 min ( P = 0.0004), and 180 min ( P = 0.0002) in VER155008 treatment group, and was observed at 60 min ( P = 0.033), 120 min ( P < 0.000087), and 180 min ( P = 0.0001) in YM-01 treatment group. ( B ) CHX chase assays of endogenous MCL-1 with or without knockdown of HSC70. The bottom graph shows the mean ± s.e.m. * P < 0.05 ( n = 3 independent experiments, shCtrl; n = 4 independent experiments, shHSC70; Welch’s t test). A significant difference was observed at 120 min ( P = 0.039) and 180 min ( P = 0.010). ( C ) CHX chase assays of endogenous MCL-1 in shHSC70 cells with or without transient FLAG-HSC70 expression. The CHX chase assay was started 48 h after transfection. The bottom graph shows the mean ± s.e.m. * P < 0.05 ( n = 3 independent experiments, respectively; Welch’s t test). A significant difference was observed at 60 min ( P = 0.026) and 120 min ( P = 0.046). ( D ) and ( E ) In vivo ubiquitination of endogenous MCL-1 ( D ) and FLAG-MCL-1 ( E ). Unmodified and polyubiquitinated FLAG-MCL-1 or endogenous MCL-1 were detected by immunoblotting with FLAG or MCL-1 antibody (*non-specific band). .

Article Snippet: Lentiviral particle production involved transfecting plasmids encoding shCtrl or shHSC70 in the pLKO.1 puromycin resistant vector or pCDH-CMV-PA-HA-CSN3-EF1 purovector into HEK293T cells together with pRSV-Rev (Addgene no. 12253), pMD 2.G (Addgene no. 12259), and pMDL g/p RRE plasmids (Addgene no. 12251) using Lipofectamine 2000.

Techniques: Inhibition, Knockdown, Expressing, Transfection, In Vivo, Ubiquitin Proteomics, Western Blot

Journal: EMBO Reports

Article Title: HSC70 coordinates COP9 signalosome and SCF ubiquitin ligase activity to enable a prompt stress response

doi: 10.1038/s44319-025-00376-x

Figure Lengend Snippet: ( A ) CHX chase assay of endogenous CDC25A with or without HSC70 knockdown, or pharmacological inhibition with YM-01. The bottom graphs show the mean ± s.e.m. * P < 0.05 ( n = 3 independent experiments; Welch’s t test). Compared with shCtrl cells or DMSO treatment, a significant difference was observed at 15 min ( P = 0.025) and 30 min ( P = 0.045) in shHSC70 cells and at 30 min ( P = 0.042) and 60 min ( P = 0.011) in YM-01 treated cells. ( B ) CHX chase assay of endogenous p27 or c-MYC with or without HSC70 knockdown. The bottom graphs show the mean ± s.e.m. * P < 0.05 ( n = 3 independent experiments, respectively; Welch’s t test). For p27 protein, a significant difference was observed at 120 min ( P = 0.032). For c-MYC protein, significant difference was observed at 60 min ( P = 0.033) and 180 min ( P = 0.045). ( A , B ) Each protein level was densitometrically quantified (normalized to 0 min) and shown in the graph below.

Article Snippet: Lentiviral particle production involved transfecting plasmids encoding shCtrl or shHSC70 in the pLKO.1 puromycin resistant vector or pCDH-CMV-PA-HA-CSN3-EF1 purovector into HEK293T cells together with pRSV-Rev (Addgene no. 12253), pMD 2.G (Addgene no. 12259), and pMDL g/p RRE plasmids (Addgene no. 12251) using Lipofectamine 2000.

Techniques: Knockdown, Inhibition

Journal: EMBO Reports

Article Title: HSC70 coordinates COP9 signalosome and SCF ubiquitin ligase activity to enable a prompt stress response

doi: 10.1038/s44319-025-00376-x

Figure Lengend Snippet: ( A ) Co-IP in HEK293T cells with or without UV irradiation (800 J/m 2 ) using HSC70 antibody, with IgG serving as a negative control. HEK293T cells were treated with 1 µM MLN4924 for 30 min immediately after UV irradiation where indicated. ( B ) Ultraviolet induced degradation of endogenous CDC25A in shCtrl and shHSC70 cells. Each protein level was densitometrically quantified (normalized to 0 min) and shown in the graph. The graphs show the mean ± s.e.m. * P < 0.05 ( n = 3 independent experiments, respectively; Welch’s t test). A significant difference was observed at 15 min ( P = 0.031) and 60 min ( P = 0.041). ( C ) Flow cytometry assessment of the cell cycle. shCtrl or shHSC70 cells were synchronized by double thymidine block and were irradiated with low dose UV irradiation (20 J/m 2 ) immediately after release. Cells were harvested and analyzed by fluorescence-assisted cell sorting (FACS) at the indicated time points after UV irradiation. ( D ) Relative proliferation rate of control or HSC70 knockdown HEK293T cells with or without UV irradiation. Relative cell counts were compared to day 0 in all graphs. The graphs show the mean ± s.e.m. * P < 0.05 ( n = 3 independent experiments, respectively; Welch’s t test). A significant difference was observed at 3 days ( P = 0.017) after UV irradiation. ( E ) Protein expression level of endogenous cleaved-PARP after UV irradiation. shCtrl or shHSC70 cells were harvested for western blotting at the indicated time point after UV irradiation. The graphs show the mean ± s.e.m. * P < 0.05 ( n = 3 independent experiments, respectively; Welch’s t test). A significant difference was observed at 6 h ( P = 0.027) and 8 h ( P = 0.048) after UV irradiation. ( F ) Caspase3/7 assay. Relative caspase 3/7 activity to those at 0 h were shown in the graph. The graphs show the mean ± s.e.m. *** P < 0.001 ( n = 3 independent experiments, respectively; Welch’s t test). A significant difference was observed at 6 h ( P = 0.0005) after UV irradiation. ( G ) Proposed interplay among HSC70, CSN, and NEDD8-SCF. Under basal conditions, HSC70 mainly interacts with CSN and enhances its deneddylation activity. Under SCF-activated conditions, HSC70 alternatively interacts with substrate-bound NEDD8-SCF, thereby promoting SCF ubiquitination activity. .

Article Snippet: Lentiviral particle production involved transfecting plasmids encoding shCtrl or shHSC70 in the pLKO.1 puromycin resistant vector or pCDH-CMV-PA-HA-CSN3-EF1 purovector into HEK293T cells together with pRSV-Rev (Addgene no. 12253), pMD 2.G (Addgene no. 12259), and pMDL g/p RRE plasmids (Addgene no. 12251) using Lipofectamine 2000.

Techniques: Co-Immunoprecipitation Assay, Irradiation, Negative Control, Flow Cytometry, Blocking Assay, Fluorescence, FACS, Control, Knockdown, Expressing, Western Blot, Activity Assay, Ubiquitin Proteomics

Journal: EMBO Reports

Article Title: HSC70 coordinates COP9 signalosome and SCF ubiquitin ligase activity to enable a prompt stress response

doi: 10.1038/s44319-025-00376-x

Figure Lengend Snippet: ( A ) Immunostaining of control or HSC70 knockdown HEK293T cells with or without low-dose UV irradiation (20 J/m 2 ) using γH2AX antibody and Hoechst 33342. Scale bars, 10 µm. ( B ) Quantification of cells with γH2AX positive foci with or without UV irradiation ( n = 6 independent experiments for shCtrl cells with or without UV, respectively; n = 7 or 5 independent experiments for shHSC70 cells with or without UV, respectively; Welch’s t test). The graphs show the mean ± s.e.m. NS not significant.

Article Snippet: Lentiviral particle production involved transfecting plasmids encoding shCtrl or shHSC70 in the pLKO.1 puromycin resistant vector or pCDH-CMV-PA-HA-CSN3-EF1 purovector into HEK293T cells together with pRSV-Rev (Addgene no. 12253), pMD 2.G (Addgene no. 12259), and pMDL g/p RRE plasmids (Addgene no. 12251) using Lipofectamine 2000.

Techniques: Immunostaining, Control, Knockdown, Irradiation

Journal: Cancers

Article Title: Estrogen Related Receptor Alpha (ERRα) a Bridge between Metabolism and Adrenocortical Cancer Progression

doi: 10.3390/cancers14163885

Figure Lengend Snippet: Metabolic changes in H295R cells related to ERRα expression levels. The metabolic profiles of H295R wild type (WT), shCTR, shERRα−/− and ERRα+/+ cells were assessed by Seahorse XFe96 Analyzer. ( a , b ) ATP Rate Assay was evaluated as indicated in “Materials and Methods”. Graphs represent the mean ± SD of three independent experiments of Total ATP Production Rate (pmol/min) ( a ) and ATP production (%) ( b ) deriving from glycolysis and oxidative phosphorylation after the sequential addition of specific inhibitors; (* p < 0.05 vs. WT). ( c – e ) Mitochondrial Stress Analysis was performed as indicated in “Materials and Methods”. Graphs represent the mean ± SD of three independent experiments of real-time oxygen consumption (OCR) rate (pmol/min/cells); (* p < 0.05 vs. shCTR). Mitochondrial Respiration ( c ), Basal Respiration ( d ), Maximal Respiration ( e ) were measured from OCR after the addition of specific inhibitors. ( f – h ) Glycolytic Stress Analysis was performed as indicated in “Materials and Methods”. Graph represents the mean ± SD of three independent experiments of Real-time extracellular acidification (ECAR) rate (mpH/min/cells); (* p < 0.05 vs. shCTR). Glycolitic function ( f ), Glycolysis ( g ) and Glycolytic Capacity ( h ) were measured from ECAR after the addition of specific inhibitors.

Article Snippet: After 48 h, cells were transfected with DNA plasmids: a plasmid encoding a scrambled short hairpin RNAs (shRNA) sequence (shCTR) (Santa Cruz, sc108060, Dallas, TX, USA), a plasmid expressing a shRNA to inhibit ERRα gene (shERRα−/−) (Santa Cruz, sc44706-sh), according to the manufacturer’s protocol (Santa Cruz Biotecnology, https://www.scbt.com/it/resources/protocols/shrna-plasmid-dna-mediated-inhibition-of-gene-expression (accessed on 7 January 2020)), or ERRα cDNA expression plasmid(ERRα+/+) (ADGENE, #10975), according to X-tremeGENETM HP DNA Transfection Reagent Protocol (Sigma).

Techniques: Expressing, Phospho-proteomics

Journal: Cancers

Article Title: Estrogen Related Receptor Alpha (ERRα) a Bridge between Metabolism and Adrenocortical Cancer Progression

doi: 10.3390/cancers14163885

Figure Lengend Snippet: ERRα modulates H295R cell motility and Vimentin expression. ( a , b ) H295R (WT), H295R clones, knock in (ERRα+/+) or knock out (shERRα−/−) for ERRα gene, and H295R cell stably transfected with control plasmid (shCTR) were used in Wound Healing ( a ) and Boyden Chamber ( b ) assays as reported in “Materials and Methods”. Images are from a representative experiment. ( c , d ) H295R cells were treated with vehicle (0) or XCT790 (1, 5, 10 μM) for 18 h and Wound Healing ( c ) and Boyden Chamber ( d ) assays were performed as reported in “Materials and Methods”. Images are from a representative experiment. ( c ) The wounds were observed under an inverted microscope immediately (0 h) and 18 h after the scratch (100× magnification). ( b , d ) Migrated cells were photographed under an inverted microscope and counted (see Material and Methods), 20× magnification. Graphs represent the mean ± SD of three independent experiments. The number of untreated cells (0) was set as 100% (* p < 0.05 vs. 0). ( e ) Total proteins from H295R clones (shCTR, shERRα−/−, ERRα+/+) were analyzed by western Blotting (WB) using antibodies against ERRα and Vimentin. GAPDH was used as a loading control. Blots are representative of three independent experiments with similar results. ( f ) H295R were transfected for 48 h with pcDNA3.1 non containing (EV) or containing ERRα coding sequence (pcDNA3.1-ERRα). After transfection cells were left untreated (−) or treated (+) for 24 h with XCT790 (10 μM). Total proteins were analyzed by WB using antibodies against ERRα and Vimentin. GAPDH was used as a loading control. ( g ) Cells were untreated (0) or treated with XCT790 (1, 5, 10 μM) for 24 h. Total proteins were analyzed by WB using antibodies against ERRα and Vimentin. GAPDH was used as a loading control. Original image of western blot can be found at .

Article Snippet: After 48 h, cells were transfected with DNA plasmids: a plasmid encoding a scrambled short hairpin RNAs (shRNA) sequence (shCTR) (Santa Cruz, sc108060, Dallas, TX, USA), a plasmid expressing a shRNA to inhibit ERRα gene (shERRα−/−) (Santa Cruz, sc44706-sh), according to the manufacturer’s protocol (Santa Cruz Biotecnology, https://www.scbt.com/it/resources/protocols/shrna-plasmid-dna-mediated-inhibition-of-gene-expression (accessed on 7 January 2020)), or ERRα cDNA expression plasmid(ERRα+/+) (ADGENE, #10975), according to X-tremeGENETM HP DNA Transfection Reagent Protocol (Sigma).

Techniques: Expressing, Clone Assay, Knock-In, Knock-Out, Stable Transfection, Transfection, Control, Plasmid Preparation, Inverted Microscopy, Western Blot, Sequencing

Journal: Cancers

Article Title: Estrogen Related Receptor Alpha (ERRα) a Bridge between Metabolism and Adrenocortical Cancer Progression

doi: 10.3390/cancers14163885

Figure Lengend Snippet: ERRα promotes H295R spheroids formation. ( a ) H295R were transfected for 48 h with pcDNA3.1 non containing (EV) or containing ERRα coding sequence (pcDNA3.1-ERRα) and then grown as 3D spheroids for 5 days. Spheroids were counted under an inverted microscope and results were expressed as fold change over control (EV) ± SD (TSFE, tumor spheroids formation efficiency); (* p < 0.05 vs. EV). Insert confirms ERRα overexpression. ( b ) H295R cells were left untreated (0) or treated with XCT790 (1, 5, 10 μM) for 24 h and TSFE was evaluated 5 days later (* p < 0.05 vs. 0). Images below graph are from a representative experiment (20× magnification). ( c ) Wild type H295R (WT) and H295R clones (shCTR, shERRα−/−, ERRα+/+) were used to evaluate 3D spheroids formation. TSFE was evaluated 5 days later (* p < 0.05 vs. WT). Images below graph are from a representative experiment (20× magnification). ( d ) H295R spheroids (H295R Sph-5) were allowed to grow for 5 days and then trypsinized and reseeded weekly in spheroid media for 5 weeks. Boyden Chamber Assay was performed as reported in the “Materials and Methods”. Migrated cells were randomly photographed and counted with ImageJ software (* p < 0.05 vs. WT). ( e ) H295R (WT) cells and H295R grown as spheroids for 5 weeks (H295R Sph-5), were analyzed by WB using antibody against Vimentin. GAPDH was used as a loading control. Blots are representative of three independent experiments with similar results. Original image of western blot can be found at .

Article Snippet: After 48 h, cells were transfected with DNA plasmids: a plasmid encoding a scrambled short hairpin RNAs (shRNA) sequence (shCTR) (Santa Cruz, sc108060, Dallas, TX, USA), a plasmid expressing a shRNA to inhibit ERRα gene (shERRα−/−) (Santa Cruz, sc44706-sh), according to the manufacturer’s protocol (Santa Cruz Biotecnology, https://www.scbt.com/it/resources/protocols/shrna-plasmid-dna-mediated-inhibition-of-gene-expression (accessed on 7 January 2020)), or ERRα cDNA expression plasmid(ERRα+/+) (ADGENE, #10975), according to X-tremeGENETM HP DNA Transfection Reagent Protocol (Sigma).

Techniques: Transfection, Sequencing, Inverted Microscopy, Control, Over Expression, Clone Assay, Boyden Chamber Assay, Software, Western Blot

Journal: Cancers

Article Title: Estrogen Related Receptor Alpha (ERRα) a Bridge between Metabolism and Adrenocortical Cancer Progression

doi: 10.3390/cancers14163885

Figure Lengend Snippet: ERRα expression levels and cholesterol influence H295R cell migration. ( a ) H295R clones (shCTR, ERRα+/+, shERRα−/−) were maintained in 5% FBS or 5% LpFS containing medium. Cells were used in Wound Healing ( a ) and Boyden Chamber ( b ) assays performed as reported in “Materials and Methods”. ( a ) Images are from a representative experiment (100× magnification). ( b ) Migrated cells were photographed under an inverted microscope (20× magnification) and counted with ImageJ software. Graphs represent the mean ± SD of three independent experiments (* p < 0.05 vs. FBS).

Article Snippet: After 48 h, cells were transfected with DNA plasmids: a plasmid encoding a scrambled short hairpin RNAs (shRNA) sequence (shCTR) (Santa Cruz, sc108060, Dallas, TX, USA), a plasmid expressing a shRNA to inhibit ERRα gene (shERRα−/−) (Santa Cruz, sc44706-sh), according to the manufacturer’s protocol (Santa Cruz Biotecnology, https://www.scbt.com/it/resources/protocols/shrna-plasmid-dna-mediated-inhibition-of-gene-expression (accessed on 7 January 2020)), or ERRα cDNA expression plasmid(ERRα+/+) (ADGENE, #10975), according to X-tremeGENETM HP DNA Transfection Reagent Protocol (Sigma).

Techniques: Expressing, Migration, Clone Assay, Inverted Microscopy, Software