Journal: Cells

Article Title: HSP90 Co-Chaperone, CacyBP/SIP, Protects α-Synuclein from Aggregation

doi: 10.3390/cells9102254

Figure Lengend Snippet: Influence of CacyBP/SIP on α-synuclein aggregation. ( A, left panel ) Representative curves showing ThT fluorescence of 30 µM α-synuclein alone (black) or in the presence of: 30 µM inactive CacyBP/SIP (grey), 0.5 µM CacyBP/SIP (light green), 30 µM CacyBP/SIP (green) or 0.5 µM HSP90 (blue). ( A, right panel ) Statistical analysis of the results for samples taken on day 4 of incubation ( n = 3). ( B, left panel ) Dot-blots showing α-synuclein immunostaining in the soluble fraction (supernatant) in samples taken on day 4 of α-synuclein aggregation, alone or in the presence of 30 µM CacyBP/SIP and ( B, right panel ) densitometric analysis of the results ( n = 3). Data, calculated as means ± SEM, are presented as percentage values. * p ≤ 0.05, *** p ≤ 0.001.

Article Snippet: The membrane was subsequently incubated in TBS-T containing 5% skim milk for 1 h. After that, the primary rabbit polyclonal anti-CacyBP/SIP antibody (Cell Signaling Technology), diluted 1:1000, or mouse anti-α-synuclein antibody (Abcam) diluted 1:1000 was applied and incubation was carried out overnight at 4 °C.

Techniques: Fluorescence, Incubation, Immunostaining

Journal: Cells

Article Title: HSP90 Co-Chaperone, CacyBP/SIP, Protects α-Synuclein from Aggregation

doi: 10.3390/cells9102254

Figure Lengend Snippet: Effect of different concentrations of CacyBP/SIP on α-synuclein aggregation. ( A, left panel ) Representative curves showing ThT fluorescence of 30 µM α-synuclein alone (black) or in the presence of: 30 µM inactive CacyBP/SIP (grey) or 30 µM (dark green), 5 µM (middle green) or 1 µM CacyBP/SIP (light green). ( A, right panel ) Statistical analysis of the results for samples taken on day 4 of incubation ( n = 3). ( B, left panel ) Dot-blots showing α-synuclein immunostaining in the soluble fraction (supernatant) of samples taken on day 4 of incubation of α-synuclein alone or in the presence of different concentrations of CacyBP/SIP and ( B, right panel ) densitometric analysis of the results ( n = 3). Data, calculated as means ± SEM, are presented as percentage values. * p ≤ 0.05, *** p ≤ 0.001.

Article Snippet: The membrane was subsequently incubated in TBS-T containing 5% skim milk for 1 h. After that, the primary rabbit polyclonal anti-CacyBP/SIP antibody (Cell Signaling Technology), diluted 1:1000, or mouse anti-α-synuclein antibody (Abcam) diluted 1:1000 was applied and incubation was carried out overnight at 4 °C.

Techniques: Fluorescence, Incubation, Immunostaining

Journal: Cells

Article Title: HSP90 Co-Chaperone, CacyBP/SIP, Protects α-Synuclein from Aggregation

doi: 10.3390/cells9102254

Figure Lengend Snippet: Effect of CacyBP/SIP on later stages of α-synuclein aggregation. ( Left panel ) Representative curves showing ThT fluorescence of 30 µM α-synuclein alone (black) or in the presence of 30 µM: inactive CacyBP/SIP (grey) or CacyBP/SIP (green) added on day 4. ( Right panel ) Statistical analysis of the results of samples taken on day 6 ( n = 3). Data, calculated as means ± SEM, are presented as percentage values.

Article Snippet: The membrane was subsequently incubated in TBS-T containing 5% skim milk for 1 h. After that, the primary rabbit polyclonal anti-CacyBP/SIP antibody (Cell Signaling Technology), diluted 1:1000, or mouse anti-α-synuclein antibody (Abcam) diluted 1:1000 was applied and incubation was carried out overnight at 4 °C.

Techniques: Fluorescence

Journal: Cells

Article Title: HSP90 Co-Chaperone, CacyBP/SIP, Protects α-Synuclein from Aggregation

doi: 10.3390/cells9102254

Figure Lengend Snippet: Influence of CacyBP/SIP on α-synuclein aggregation assessed by transmission electron microscopy (TEM). Representative micrographs of α-synuclein obtained before initiation of aggregation ( A ), on day 4 of incubation ( B ), on day 4 of incubation in the presence of: 30 µM inactive CacyBP/SIP ( C ), 15 µM CacyBP/SIP ( D ) or 30 µM CacyBP/SIP ( E ). Scale bar—200 nm (A–D) and 100 nm ( E ).

Article Snippet: The membrane was subsequently incubated in TBS-T containing 5% skim milk for 1 h. After that, the primary rabbit polyclonal anti-CacyBP/SIP antibody (Cell Signaling Technology), diluted 1:1000, or mouse anti-α-synuclein antibody (Abcam) diluted 1:1000 was applied and incubation was carried out overnight at 4 °C.

Techniques: Transmission Assay, Electron Microscopy, Incubation

Journal: Cells

Article Title: HSP90 Co-Chaperone, CacyBP/SIP, Protects α-Synuclein from Aggregation

doi: 10.3390/cells9102254

Figure Lengend Snippet: Influence of CacyBP/SIP domains on α-synuclein aggregation. ( A, left panel ) Representative curves showing ThT fluorescence of 30 µM α-synuclein alone (black) or in the presence of 30 µM: inactive CacyBP/SIP (grey), CacyBP/SIP (green), N domain (orange), CS domain (yellow) or SGS domain (brown). ( A, right panel ) Statistical analysis of the results ( n = 3). ( A’, left panel ) Dot-blots showing α-synuclein immunostaining in the soluble fraction and ( A’, right panel ) statistical analysis of the results ( n = 3). (B, left panel) Representative curves showing ThT fluorescence of 30 µM α-synuclein alone (black) or in the presence of 30 µM: inactive CacyBP/SIP (grey), CacyBP/SIP (green) or NCS fragment of CacyBP/SIP (violet) ( B, right panel ) Statistical analysis of the results ( n = 3). ( B’, left panel ) Dot-blots showing α-synuclein immunostaining in the soluble fraction and ( B’, right panel ) statistical analysis of the results ( n = 3). In all cases, samples were taken on day 4 of incubation and data, calculated as means ± SEM, are presented as percentage values. ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: The membrane was subsequently incubated in TBS-T containing 5% skim milk for 1 h. After that, the primary rabbit polyclonal anti-CacyBP/SIP antibody (Cell Signaling Technology), diluted 1:1000, or mouse anti-α-synuclein antibody (Abcam) diluted 1:1000 was applied and incubation was carried out overnight at 4 °C.

Techniques: Fluorescence, Immunostaining, Incubation

Journal: Cells

Article Title: HSP90 Co-Chaperone, CacyBP/SIP, Protects α-Synuclein from Aggregation

doi: 10.3390/cells9102254

Figure Lengend Snippet: Interaction between α-synuclein and CacyBP/SIP studied by ELISA ( A ) and chemical crosslinking ( B ). ( A ) Upper panel shows absorbance measured at different molar ratio of CacyBP/SIP (concentration 0, 3.89, 7.78, 19.45, 31.12, and 58.35 µM) to α-synuclein (3.89 µM) while the lower one shows quantitative analysis of the results obtained from 3 independent experiments. Data are presented as means ± standard errors (SEM); * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. ( B ) 15% polyacrylamide gel stained with Coomassie brilliant blue R250. α-synuclein (30 μM) was mixed with CacyBP/SIP (30 μM) Lane 1 and 4, α-synuclein alone; Lanes 2 and 5, mixture of α-synuclein and CacyBP/SIP; Lanes 3 and 6, CacyBP/SIP alone. Proteins were incubated with (lanes 1–3) and without crosslinking agent (lanes 4–6) and then 15 µl of reaction mixture was applied on the gel. “*” indicates the α-synuclein-CacyBP/SIP crosslinking product. A representative experiment, out of 3 performed, is shown.

Article Snippet: The membrane was subsequently incubated in TBS-T containing 5% skim milk for 1 h. After that, the primary rabbit polyclonal anti-CacyBP/SIP antibody (Cell Signaling Technology), diluted 1:1000, or mouse anti-α-synuclein antibody (Abcam) diluted 1:1000 was applied and incubation was carried out overnight at 4 °C.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Staining, Incubation

Journal: Cells

Article Title: HSP90 Co-Chaperone, CacyBP/SIP, Protects α-Synuclein from Aggregation

doi: 10.3390/cells9102254

Figure Lengend Snippet: Presence of complexes formed between α-synuclein and CacyBP/SIP or HSP90 in HEK293 cells visualized by PLA (representative images). Complexes of examined proteins are shown as red dots; cell nuclei, stained with DAPI, are in blue. HSP90 was used as a positive control. Scale bar–10 μm.

Article Snippet: The membrane was subsequently incubated in TBS-T containing 5% skim milk for 1 h. After that, the primary rabbit polyclonal anti-CacyBP/SIP antibody (Cell Signaling Technology), diluted 1:1000, or mouse anti-α-synuclein antibody (Abcam) diluted 1:1000 was applied and incubation was carried out overnight at 4 °C.

Techniques: Staining, Positive Control

Journal: Cells

Article Title: HSP90 Co-Chaperone, CacyBP/SIP, Protects α-Synuclein from Aggregation

doi: 10.3390/cells9102254

Figure Lengend Snippet: Influence of CacyBP/SIP on the number of α-synuclein aggregates in HEK293 cells. ( A ) A scheme showing preparation of α-synuclein seeds and their delivery to HEK293 cells. ( B, upper part ) Representative immunofluorescence staining performed with the use of primary conformation-specific anti-α-synuclein antibody. Aggregates/inclusions of α–synuclein are visible in green. Insert shows enlargement of inclusion. ( B, lower part ) Statistical analysis of the results from 3 independent experiments (30 inclusion-containing cells were analyzed) are presented as means ± standard errors (SEM); * p ≤ 0.05. Scale bar—5 μm.

Article Snippet: The membrane was subsequently incubated in TBS-T containing 5% skim milk for 1 h. After that, the primary rabbit polyclonal anti-CacyBP/SIP antibody (Cell Signaling Technology), diluted 1:1000, or mouse anti-α-synuclein antibody (Abcam) diluted 1:1000 was applied and incubation was carried out overnight at 4 °C.

Techniques: Immunofluorescence, Staining

Journal: Cells

Article Title: HSP90 Co-Chaperone, CacyBP/SIP, Protects α-Synuclein from Aggregation

doi: 10.3390/cells9102254

Figure Lengend Snippet: Viability of HEK293 cells overexpressing CacyBP/SIP after rotenone treatment. Cells treated with solvent (black bar), cells transfected with 3xFLAG (white bar) or with 3xFLAG-CacyBP/SIP (grey bar) treated with rotenone. Cell viability was quantified using an MTS assay. Data obtained from 3 independent experiments, calculated as means ± SEM, are presented as percentage values. * p ≤ 0.05.

Article Snippet: The membrane was subsequently incubated in TBS-T containing 5% skim milk for 1 h. After that, the primary rabbit polyclonal anti-CacyBP/SIP antibody (Cell Signaling Technology), diluted 1:1000, or mouse anti-α-synuclein antibody (Abcam) diluted 1:1000 was applied and incubation was carried out overnight at 4 °C.

Techniques: Transfection, MTS Assay

Journal: Gland Surgery

Article Title: Long noncoding RNA SNHG6 promotes papillary thyroid cancer cells proliferation via regulating miR-186/CDK6 axis

doi: 10.21037/gs-21-586

Figure Lengend Snippet: SNHG6 is highly expressed in PTC (A,B) The UALCAN database was used to analyze the expression of SNHG6 in THCA and normal tissues and its correlation with tumor stage. (C) Expression of SNHG6 in PTC and Nthy-ori 3-1 cell was analyzed by qRT-PCR. (D,E) qRT-PCR verification of SNHG6 overexpression and silencing in PTC cells. *, P<0.05. SNHG6, small nucleolar RNA host gene 6; PTC, papillary thyroid cancer; THCA, thyroid carcinoma; qRT-PCR, quantitative real-time PCR; TCGA, The Cancer Genome Atlas; NC, normal control.

Article Snippet: Nthy-ori 3-1 and PTC cell lines (TPC-1 and K1) were purchased from American Type Culture Collection (VA, USA).

Techniques: Expressing, Quantitative RT-PCR, Over Expression, Real-time Polymerase Chain Reaction

Journal: Gland Surgery

Article Title: Long noncoding RNA SNHG6 promotes papillary thyroid cancer cells proliferation via regulating miR-186/CDK6 axis

doi: 10.21037/gs-21-586

Figure Lengend Snippet: miR-186 as the downstream target of SNHG6. (A) Binding sites of SNHG6 and miR-186 were predicted by the Starbase database. (B) Luciferase reporter gene assay was used to verify the binding sites of SNHG6 and miR-186. (C) Expression of miR-186 in thyroid cancer and normal tissues analyzed by the UALCAN database. (D) Expression of miR-186 in PTC cells and Nthy-ori 3-1 cells analyzed by qRT-PCR. *, P<0.05. SNHG6, small nucleolar RNA host gene 6; PTC, papillary thyroid cancer; qRT-PCR, quantitative real-time PCR; WT, wild-type; MUT, mutation; NC, normal control; THCA, thyroid carcinoma; TCGA, The Cancer Genome Atlas.

Article Snippet: Nthy-ori 3-1 and PTC cell lines (TPC-1 and K1) were purchased from American Type Culture Collection (VA, USA).

Techniques: Binding Assay, Luciferase, Reporter Gene Assay, Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Mutagenesis

Journal: Gland Surgery

Article Title: Long noncoding RNA SNHG6 promotes papillary thyroid cancer cells proliferation via regulating miR-186/CDK6 axis

doi: 10.21037/gs-21-586

Figure Lengend Snippet: SNHG6 regulates PTC cell proliferation through miR-186. (A,B) CCK-8 experiment was used to study the effect of SNHG6 regulating miR-186 on the activity of PTC cells. (C,D) Colony formation assay to study the effect of SNHG6 regulating miR-186 on the colony formation of PTC cells. Cells were stained with 0.5% crystal violet solution. (E,F) EdU assay was used to study the effect of SNHG6 on the proliferation of PTC cells (magnification 100×). *, P<0.05. SNHG6, small nucleolar RNA host gene 6; PTC, papillary thyroid cancer; CCK-8, cell counting kit-8; EdU, 5-ethynyl-2’-deoxyuridine; OD, optical density; NC, normal control; DAPI, 4’,6-diamidino-2-phenylindole.

Article Snippet: Nthy-ori 3-1 and PTC cell lines (TPC-1 and K1) were purchased from American Type Culture Collection (VA, USA).

Techniques: CCK-8 Assay, Activity Assay, Colony Assay, Staining, EdU Assay, Cell Counting

Journal: Cancer Cell International

Article Title: RRS1 gene expression involved in the progression of papillary thyroid carcinoma

doi: 10.1186/s12935-018-0519-x

Figure Lengend Snippet: Knock-down of RRS1 inhibited TPC-1 cells proliferation. a , b Real-time PCR and Western blotting were performed to detect the efficiency of RRS1 knock-down. c Proliferation of TPC-1 cells was expressing shRNA targeted against negative and RRS1. Plotted data were mean ± s.e.m. of three independent experiments. * P < 0.05 from student T-test. d Colony formation assays of TPC-1 cells expressing shRNAs were quantified for relative colony numbers of three independent experiments. * P < 0.05 from student T-test. Three independent experiments were performed

Article Snippet: Human papillary thyroid carcinoma cell line TPC-1 was obtain from American Type Culture Collection.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, shRNA

Journal: Cancer Cell International

Article Title: RRS1 gene expression involved in the progression of papillary thyroid carcinoma

doi: 10.1186/s12935-018-0519-x

Figure Lengend Snippet: Knock-down of RRS1 induced G 2 /M phase arrest and apoptosis in TPC-1 cells. a Left: one representative flow cytometry analysis of the cell cycle; right: histograms showed the percentage of cell subpopulations in G 1 , S, and G 2 /M phases. Data represent mean ± SE (n = 3). b Left: one representative flow cytometry analysis of the Annexin-V; right: histograms showed the percentage of cell subpopulations in M1 gate (Annexin-V positive cells). Data represent mean ± SE (n = 3). Three independent experiments were performed. * P value < 0.1, ** P value < 0.01, *** P value < 0.001

Article Snippet: Human papillary thyroid carcinoma cell line TPC-1 was obtain from American Type Culture Collection.

Techniques: Flow Cytometry