filter trap analysis  (GE Healthcare)

Journal: Acta neuropathologica

Article Title: Insulin‑like growth factor 2 (IGF2) protects against Huntington’s disease through the extracellular disposal of protein aggregates

doi: 10.1007/s00401-020-02183-1

Figure Lengend Snippet: expression reduces the levels of polyQ79 aggregates and HMW species. a Neuro2a cells were co-transfected with a polyQ79-EGFP expression vector and IGF2 plasmid or empty vector (Mock). 24 or 48 h later, the number of polyQ79-EGFP inclusions were visualized by fluorescence microscopy (left panel). Quantification of inclusions was normalized by the number of GFP-positive cells (right panel; n = 100 to 350 cells per experiment). Scale bar, 20 μm. b PolyQ79-EGFP expression was analyzed in whole cell extracts by western blot analysis using an anti-GFP antibody. A non-tagged version of IGF2 expression vector was also included. The presence of high molecular weight (HMW) species is indicated. Hsp90 and IGF2 levels were also determined (left panel). PolyQ79-EGFP HMW species levels were quantified and normalized to Hsp90 levels (right panel). c Filter trap assay was performed in the same cell extracts analyzed in b (left panel) and quantified at 24 h (right panel). d Neuro2a cells were transiently co-transfected with polyQ79-EGFP together with plasmids to express IGF1, IGF2 or empty vector (Mock). The presence of polyQ79-EGFP HMW species was analyzed in whole cell extracts after 24 h by western blot analysis. Hsp90 expression was monitored as loading control. e Neuro2a cells were co-transfected with polyQ79-EGFP or SOD1G85R-GFP and IGF2 plasmid or empty vector (Mock). After 24 h, polyQ79 and SOD1 HMW species were measured in total cell extracts using western blot under non-reducing conditions (without DTT). Hsp90 expression was monitored as loading control. In all quantifications, values represent the mean and SEM of at least three independent experiments. Statistically significant differences detected by two-tailed unpaired t test (***p < 0.001; **p < 0.01; *p < 0.05)

Article Snippet: Microscopy, western blot and filter trap analysis Neuro2a and HEK293T cells were obtained from ATCC and maintained in Dulbecco’s modified Eagles medium supplemented with 5% fetal bovine serum and Penicillin/Streptomycin (Gibco).

Techniques: Expressing, Transfection, Plasmid Preparation, Fluorescence, Microscopy, Western Blot, High Molecular Weight, TRAP Assay, Control, Two Tailed Test

Journal: Acta neuropathologica

Article Title: Insulin‑like growth factor 2 (IGF2) protects against Huntington’s disease through the extracellular disposal of protein aggregates

doi: 10.1007/s00401-020-02183-1

Figure Lengend Snippet: treatment reduces the accumulation of polyQ79 and mHttQ85 HMW species. a Neuro2a cells were transfected with polyQ79-EGFP expression vectors in the presence of IGF2-enriched cell culture media. 24 h later, polyQ79-EGFP and mHttQ85-GFP inclusions were visualized by microscopy (upper panel) and quantified (bottom panel; n = 100 to 350 cells per experiment). Scale bar 20 μm. b Neuro2a cells were transfected with polyQ79-EGFP or mHttQ85-GFP expression vectors in the presence of IGF2-enriched cell culture media. 24 h later, the HMW species levels of polyQ79-EGFP or mHttQ85-GFP were determined by western blot analysis using anti-GFP antibody. Hsp90 expression was monitored as loading control. c Filter trap assay was performed using the same protein extracts analyzed in b to quantify polyQ79-EGFP and mHttQ85-GFP aggregates. d Neuro2a cells were transfected with polyQ79-EGFP in the presence of 1 μM insulin. 24 h later, the HMW species levels of polyQ79-EGFP protein were measured in total cell extracts by western blot. Hsp90 expression was monitored as loading control. e Neuro2a cells were transfected with polyQ79-EGFP or mHttQ85-GFP expression vectors. After 24 h, cells were treated with IGF2-enriched cell culture media and 24 h later, HMW species levels were determined and quantified by western blot analysis using anti-GFP antibody. Hsp90 levels were monitored as loading control. f Filter trap assay was performed in the same cell extracts analyzed in (e) and polyQ79-EGFP, and mHttQ85-GFP aggregates were detected using anti-GFP antibody. In all quantifications, average and SEM of at least three independent experiments are shown. Statistically significant differences detected by two-tailed unpaired t test (***p < 0.001; **p < 0.01; *p < 0.05)

Article Snippet: Microscopy, western blot and filter trap analysis Neuro2a and HEK293T cells were obtained from ATCC and maintained in Dulbecco’s modified Eagles medium supplemented with 5% fetal bovine serum and Penicillin/Streptomycin (Gibco).

Techniques: Transfection, Expressing, Cell Culture, Microscopy, Western Blot, Control, TRAP Assay, Two Tailed Test

Journal: Acta neuropathologica

Article Title: Insulin‑like growth factor 2 (IGF2) protects against Huntington’s disease through the extracellular disposal of protein aggregates

doi: 10.1007/s00401-020-02183-1

Figure Lengend Snippet: Stimulation of cells with IGF2 reduces the half-life of mHtt. a HEK293T cells were co-transfected with mHttQ43-GFP expression vector with IGF2 plasmid (middle panel) or empty vector (Mock) (upper panel) and then pulse labeled with 35S for indicated time points. Autoradiography (AR) indicated the 35S signal for each time point. Data were quantified and normalized to time point 0 h (lower panel). b Similar experiments as described in a were monitored for additional time points. Data were quantified and normalized to the time point 1 h (lower panel). c HEK293T cells were transfected with mHttQ43-GFP and IGF2 expression vectors. Pulse was performed 24 h after transfection. Cells were treated with 30 μM chloroquine (CQ) or 1 μM bortezomib (Bort) at the beginning of the chasing for additional 21 h (upper panel). Data were quantified and normalized to time point 1 h (lower panel). d Neuro2a cells were co-transfected with a polyQ79-EGFP expression vector and IGF2 plasmid or empty vector (Mock) for 8 h and then treated with 1 μM lactacystin (Lact) for additional 16 h (upper panel). Quantification of inclusions per GFP-positive cell was performed (lower panel; n = 100 to 350 cells per experiment). e Neuro2a cells were co-transfected with a polyQ79-EGFP expression vector and IGF2 plasmid or empty vector (Mock) for 8 h and then treated with 1 μM lactacystin (Lact) or 1 μM MG132 for additional 16 h. PolyQ79-EGFP HMW species were analyzed in whole cells extracts by western blot using anti-GFP antibody and quantified (lower panel). Hsp90 expression was analyzed as loading control (upper panel). f Neuro2a cells were co-transfected with a polyQ79-EGFP expression vector and IGF2 plasmid or empty vector (Mock) for 8 h and then treated with 30 μM chloroquine (CQ) for additional 16 h. Endogenous lipidated LC3-II levels were monitored by western blot using anti-LC3 antibody. Hsp90 expression was monitored as loading control (upper panel). LC3 II levels were quantified and normalized to Hsp90 (lower panel). g Neuro2a cells were co-transfected with polyQ79-EGFP and IGF2 expression vectors or empty vector (Mock) for 8 h and then treated with 30 μM CQ for additional 16 h (upper panel). Quantification of inclusions per GFP-positive cell was performed (lower panel; n = 100 to 350 cells per experiment). h Neuro2a cells were co-transfected with polyQ79-EGFP and IGF2 expression vectors or empty vector (Mock) for 8 h and then treated with 30 μM CQ for additional 16 h. PolyQ79-EGFP HMW species were analyzed in whole cell extracts by western blot using anti-GFP antibody and quantified (lower panel). Hsp90 expression was monitored as loading control (upper panel). i Filter trap was performed using the same cells extracts analyzed in (h). In all quantifications, average and SEM of at least three independent experiments are shown. Statistically significant differences detected by two-tailed unpaired t test (***p < 0.001; **p < 0.01; *p < 0.05)

Article Snippet: Microscopy, western blot and filter trap analysis Neuro2a and HEK293T cells were obtained from ATCC and maintained in Dulbecco’s modified Eagles medium supplemented with 5% fetal bovine serum and Penicillin/Streptomycin (Gibco).

Techniques: Transfection, Expressing, Plasmid Preparation, Labeling, Autoradiography, Western Blot, Control, Two Tailed Test

Journal: Acta neuropathologica

Article Title: Insulin‑like growth factor 2 (IGF2) protects against Huntington’s disease through the extracellular disposal of protein aggregates

doi: 10.1007/s00401-020-02183-1

Figure Lengend Snippet: enhances the extracellular release of polyQ79 and mHttQ85 through unconventional secretion. a Neuro2a cells were co-transfected with polyQ79-EGFP or SOD1G85R plasmid and IGF2 plasmid or empty vector (Mock). After 16 h, cell culture media was replaced for Optimem and then collected after 24 h for dot blot or b filter trap analysis. c Neuro2a cells were transfected with siRNAs against InsR or IGF1R mRNAs. After 24 h, cells were co-transfected with polyQ79-EGFP and IGF2 expression vector or empty vector (Mock). Then, cell culture media was replaced for Optimem for 24 h. In addition, an anti-IGF2R was added when indicated. The presence of polyQ79-EGFP in the cell culture media analyzed by dot blot using an anti-GFP antibody and quantified. The knockdown of indicated proteins was confirmed in cell extracts using semiquantitative PCR (right panel). d Neuro2a cells were co-transfected with polyQ79-EGFP and IGF2 plasmid or empty vector (Mock). Then, cell culture media was replaced for Optimem for 8 h and treated with 2 μM brefeldin A (Bref A) or vehicle for additional 16 h. Cell culture media was collected and analyzed by dot blot using an anti-GFP antibody (upper panel) and quantified (lower panel). Image was cropped from the same membrane and film exposure. e Cells described in d were analyzed by western blot using anti-GFP antibody. Hsp90 expression was monitored as loading control. PolyQ79-EGFP levels were quantified and normalized to Hsp90 (lower panel). f Extracellular vesicles from Neuro2a were analyzed by NanoSight nanotracking analysis and plotted by size (upper panel) and total concentration (lower panel) in cells expressing or not with IGF2. g Extracellular vesicles from Neuro2a cells co-transfected with polyQ79-EGFP and IGF2 expression vectors or empty vector (Mock) were concentrated followed by western blot analysis. h Isolated microvesicles (MV) and exosomes, together with total cell extracts from Neuro2a cells co-transfected with polyQ79-EGFP and IGF2 expression vectors or empty vector (Mock), were analyzed by western blot. In all quantifications, average and SEM of at least three independent experiments are shown. Statistically significant differences detected by two-tailed unpaired t test (***p < 0.001; **p < 0.01; *p < 0.05)

Article Snippet: Microscopy, western blot and filter trap analysis Neuro2a and HEK293T cells were obtained from ATCC and maintained in Dulbecco’s modified Eagles medium supplemented with 5% fetal bovine serum and Penicillin/Streptomycin (Gibco).

Techniques: Transfection, Plasmid Preparation, Cell Culture, Dot Blot, Expressing, Knockdown, Membrane, Western Blot, Control, Concentration Assay, Isolation, Two Tailed Test

Journal: Acta neuropathologica

Article Title: Insulin‑like growth factor 2 (IGF2) protects against Huntington’s disease through the extracellular disposal of protein aggregates

doi: 10.1007/s00401-020-02183-1

Figure Lengend Snippet: signaling induces the release of polyQ79 through cytoskeleton remodeling. a Quantitative proteomics was performed in protein extracts derived from Neuro2a cells transiently transfected with IGF2 or empty vector (Mock) for 24 h. Data were analyzed and plotted in a volcano graph as fold change. Vertical dashed lines indicate a log2 > 0.1 of fold change. Proteins related to actin cytoskeleton function are highlighted in green. Dots in blue represent associated proteins to actin cytoskeleton with a log2 < 0.1 fold change. b Functional enrichment analysis. Blue scale refers to the number of proteins that are known to be involved in each pathway. Red scale refers to the number of proteins altered in IGF2 overexpressing cells for each pathway. c Neuro2a cells were transfected with a plasmid encoding Life-Actin to monitor actin cytoskeleton dynamics. Cells were plated onto fibronectin-coated plates and recorded by time-lapse confocal microscopy every 40 s for 5 min. Time-lapse microscopy was performed after treatment with IGF2-enriched media (left panel). Quantification of cortical and internal actin clusters is shown (right panel). d Phalloidin-rhodamine staining of MEF cells after 5 min of treatment with IGF2-enriched media. Scale bar, 50 μm (right panel). Quantification of actin clusters is presented (left panel). e Neuro2a cells were treated with IGF2-enriched media or control media derived from Mock transfected cells. After 5 min, cells were lysed and cell extracts prepared to measure Rac1-GTP levels by pull-down assay. f Neuro2a cells were transfected with RacN17 or RacV12 or empty vector (Mock). 24 h later, cells were co-transfected with expression vectors for polyQ79-EGFP and IGF2 or empty vector (Mock) and then incubated with Optimem. The presence of polyQ79-EGFP in the cell culture media was determined using dot blot (upper panel) and quantified (lower panel). g Neuro2a cells were co-transfected with expression vectors for polyQ79-EGFP and IGF2 or empty vector (Mock). Then, cell culture media was replaced for Optimem for 8 h and treated with 100 μM NSC23766 (NSC) for additional 16 h. Cell culture media was collected and analyzed by dot blot using an anti-GFP antibody (upper panel) and quantified (lower panel). Image was cropped from the same membrane and film exposure. h Neuro2a cells were co-transfected with polyQ79-EGFP plasmid and IGF2 plasmid or empty vector (Mock) for 8 h and then treated with 100 μM NSC23766 (NSC) or vehicle for additional 16 h. PolyQ79-EGFP HMW species were analyzed in cell lysates by western blot using anti-GFP antibody. Hsp90 expression was monitored as loading control (left panel). PolyQ79-EGFP levels were quantified and normalized to Hsp90 levels (right panel). In all quantifications, average and SEM of at least three independent experiments are shown. Statistically significant differences detected by two-tailed unpaired t test (***p < 0.001; *p < 0.05)

Article Snippet: Microscopy, western blot and filter trap analysis Neuro2a and HEK293T cells were obtained from ATCC and maintained in Dulbecco’s modified Eagles medium supplemented with 5% fetal bovine serum and Penicillin/Streptomycin (Gibco).

Techniques: Quantitative Proteomics, Derivative Assay, Transfection, Plasmid Preparation, Functional Assay, Confocal Microscopy, Time-lapse Microscopy, Staining, Control, Pull Down Assay, Expressing, Incubation, Cell Culture, Dot Blot, Membrane, Western Blot, Two Tailed Test