Journal: Biochemistry

Article Title: Humanin Blocks the Aggregation of Amyloid- β Induced by Acetylcholinesterase, an Effect Abolished in the Presence of IGFBP-3

doi: 10.1021/acs.biochem.0c00274

Figure Lengend Snippet: Increasing concentrations of IGFBP-3 bind HN and abolish its binding to Aβ in the absence or presence of AChE. (A) Interaction of Aβ40 (■) or Aβ42 (●) with biotinylated HN. Aβ (100 nM) was bound to the wells. Increasing concentrations of biotinylated HN were then added and processed as described in the experimental procedures. Optical density measurements (450 nm) were normalized by expressing each point in relation to the best-fit Emax value (set to 100%). The data were then plotted as a function of the increasing biotinylated HN concentrations. Using GraphPad Prism 8.3.1, the data were fit to a single binding site model with a nonlinear regression curve fitting approach and plotted as the mean ± SD of three independent trials, each of which was run in triplicate. (B and C) Competition of IGFBP-3 for the complex between HN and Aβ40 or Aβ42. Aβ (100 nM) was bound to the plate wells. Then, a single concentration of biotinylated HN (100 nM, ●) or a combination of HN and AChE [biotinylated HN (100 nM) + AChE (100 nM)] (■) was incubated for 1 h with or without increasing concentrations of IGFBP-3 and loaded into the Aβ coated wells. The signal was then processed as described in the Experimental Procedures. Arrows on the x-axis indicate the IGFBP-3 concentration corresponding to 50% inhibition for each of the following curves: Aβ40 (●, 93 ± 16 nM), Aβ40 (■, 74 ± 14 nM), Aβ42 (●, 58 ± 11 nM), and Aβ42 (■, 39 ± 7 nM). The dashed line indicates 50% of the maximum binding. Prior to data analysis, the OD was corrected for nonspecific binding by subtracting the mean background absorbance for the negative controls prepared with all components except biotinylated HN. Data were then normalized by plotting the mean absorbances for each concentration as a fraction of the maximal binding (set to 100%) and as a function of the IGFBP-3 concentrations. Using GraphPad Prism 8.3.1, the data were analyzed with a nonlinear regression curve fitting approach then expressed as the mean ± SD of three independent experiments, each of which was carried out in triplicate.

Article Snippet: The graphs (E–G) were prepared using the GraphPad 8.3.1 software and summarize the results expressed as the mean ± SD.

Techniques: Binding Assay, Expressing, Concentration Assay, Incubation, Inhibition

Journal: Biochemistry

Article Title: Humanin Blocks the Aggregation of Amyloid- β Induced by Acetylcholinesterase, an Effect Abolished in the Presence of IGFBP-3

doi: 10.1021/acs.biochem.0c00274

Figure Lengend Snippet: IGFBP-3, but not AChE, blocks the binding of HN and Aβ. (A) Interaction of AChE with Aβ40 (■) or Aβ42 (●). AChE (100 nM) was bound to the plate wells. Increasing concentrations of biotinylated Aβ were then added to the wells and processed as described in the Experimental Procedures. Optical densities (450 nm) were normalized for both curves by expressing each point relative to the best fitted Emax value (set to 100%). The data were then plotted as a function of the increasing biotinylated Aβ concentrations and, using GraphPad Prism 8.3.1, fit to a single binding site model with a nonlinear regression curve fitting approach. Data were expressed as the mean ± SD of three independent experiments, each of which was carried out in triplicate. (B and C) HN or Aβ (0.2 μM) was bound to the wells. Biotinylated Aβ (0.2 μM) or biotinylated HN (0.2 μM) was then pre-incubated with increasing concentrations of AChE for 1 h at rt and then added to the wells in the absence or presence of IGFBP-3 (0.2 μM or 2 μM). For samples without IGFBP-3, the optical density measurements were normalized by plotting the mean absorbances for each concentration as a fraction of the maximal binding (set to 100%), followed by plotting the data as a function of AChE concentrations. The negative control had the same concentrations of HN or Aβ and AChE, but water was substituted in place of biotinylated Aβ or biotinylated HN. For samples with IGFBP-3, the optical density was expressed as percent of the corresponding sample without IGFBP-3. Data were presented as the mean ± SD of three independent assays.

Article Snippet: The graphs (E–G) were prepared using the GraphPad 8.3.1 software and summarize the results expressed as the mean ± SD.

Techniques: Binding Assay, Expressing, Incubation, Concentration Assay, Negative Control

Journal: Biochemistry

Article Title: Humanin Blocks the Aggregation of Amyloid- β Induced by Acetylcholinesterase, an Effect Abolished in the Presence of IGFBP-3

doi: 10.1021/acs.biochem.0c00274

Figure Lengend Snippet: HN reduced Aβ aggregation, an effect reversed upon the addition of IGFBP-3. Pretreated monomeric Aβ (2 μM) was incubated with 2 μM HN in the absence or presence of 2 or 6 μM IGFBP-3 ((A) Aβ40, AS-72215 and (B) Aβ42, AS-72216). Thioflavin fluorescence (Ex 440 nm, Em 484 nm) was monitored at 37 °C for 335 min every 2.5 min, with 15 s of shaking between the readings. Assay buffer alone, IGFBP-3, or HN was used as a blank. Each measurement was corrected for the baseline fluorescence in the absence of Aβ. The change in fluorescence units (RFU) relative to the first measurement at t0 is shown. The data were normalized to the maximum ThT level and plotted using GraphPad Prism 8.3.1.

Article Snippet: The graphs (E–G) were prepared using the GraphPad 8.3.1 software and summarize the results expressed as the mean ± SD.

Techniques: Incubation, Fluorescence

Journal: Biochemistry

Article Title: Humanin Blocks the Aggregation of Amyloid- β Induced by Acetylcholinesterase, an Effect Abolished in the Presence of IGFBP-3

doi: 10.1021/acs.biochem.0c00274

Figure Lengend Snippet: HN blocks the aggregation of Aβ induced by the addition of AChE, an effect abolished upon the addition of IGFBP-3. Pretreated monomeric Aβ (2 μM) was incubated with 2 μM AChE, 2 μM HN, or both in the absence or presence of 2 or 6 μM IGFBP-3 ((A) Aβ40, AS-72215 and (B) Aβ42, AS-72216). Assay buffer alone, HN, or IGFBP-3 was used as a blank. Thioflavin fluorescence (Ex 440 nm, Em 484 nm) was monitored at 37 °C for 335 min every 2.5 min, with 15 s of shaking between readings. Each measurement was corrected for the baseline fluorescence in the absence of Aβ. The change of the RFU in relation to the first measurement at t0 is shown. The data were normalized to the maximum ThT level and plotted using GraphPad Prism 8.3.1.

Article Snippet: The graphs (E–G) were prepared using the GraphPad 8.3.1 software and summarize the results expressed as the mean ± SD.

Techniques: Incubation, Fluorescence

Journal: Biochemistry

Article Title: Humanin Blocks the Aggregation of Amyloid- β Induced by Acetylcholinesterase, an Effect Abolished in the Presence of IGFBP-3

doi: 10.1021/acs.biochem.0c00274

Figure Lengend Snippet: Both AChE and HN were found in a complex with Aβ from the medium of A549 cells using 6E10 antibodies; however, the addition of an exogenous HN peptide reduced but did not abolish the binding of AChE to Aβ. Anti-Aβ specific antibodies (6E10) were added (1:1000 dilution) to the ELISA wells. The wells were blocked, and 300 μL of the conditioned medium (0.5 μg/μL of A549 cells or H1299 cells, 72 h after serum starvation) was added. The amount of HN and AChE bound was detected using the corresponding specific primary antibodies and then processed as described in the Experimental Procedures. The fold change relative to the controls incubated with all components except the primary antibodies was calculated and fit with a nonlinear regression curve using GraphPad Prism 8.3.1. The data represent the mean ± SD of three separate experiments, each of which was performed in triplicate.

Article Snippet: The graphs (E–G) were prepared using the GraphPad 8.3.1 software and summarize the results expressed as the mean ± SD.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Biochemistry

Article Title: Humanin Blocks the Aggregation of Amyloid- β Induced by Acetylcholinesterase, an Effect Abolished in the Presence of IGFBP-3

doi: 10.1021/acs.biochem.0c00274

Figure Lengend Snippet: HN was detected upon using Aβ 6E10 antibodies, while different levels of AChE were detected using all the amyloid antibodies. Cells (0.2 × 105 cells per well) were seeded in 96-well plates in a 10% FBS-supplemented medium. The next day, the cells were incubated in a serum-free medium for 72 h. Specific antibodies were added (1:1000 dilution) to the ELISA wells. After blocking the wells, 300 μL of the A549 cell conditioned medium (0.5 μg/μL, 72 h post serum starvation) was added. The proteins and peptides were detected using their corresponding primary antibodies and then processed as described in the Experimental Procedures. Each column represents the mean ± SD of three independent and separate experiments, each of which was performed in triplicate. Data processing was carried out using the GraphPad 8.3.1 software. Asterisks (*) indicate a statistically significant difference between each treatment relative to the mIgG controls. The absence of asterisks indicates no significance (Mann–Whitney test, *p < 0.05, **p < 0.01).

Article Snippet: The graphs (E–G) were prepared using the GraphPad 8.3.1 software and summarize the results expressed as the mean ± SD.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Blocking Assay, Software, MANN-WHITNEY

Journal: Biochemistry

Article Title: Humanin Blocks the Aggregation of Amyloid- β Induced by Acetylcholinesterase, an Effect Abolished in the Presence of IGFBP-3

doi: 10.1021/acs.biochem.0c00274

Figure Lengend Snippet: The concentration of HN found in a complex using 6E10 antibodies is reduced upon the addition of IGFBP-3 and correlates with an increase in the amount of amyloid oligomers. Anti-Aβ specific antibodies (6E10) were added (1:1000 dilution) to the ELISA wells. The wells were blocked, and then 300 μL of the conditioned medium (0.5 μg/μL of A549 cells or H1299 cells, 72 h after serum starvation) was added in the absence or presence of increasing IGFBP-3 concentrations. IGFBP-3, HN, amyloid oligomers, and fibrils were detected using the corresponding specific primary antibodies and then processed as described in the Experimental Procedures. The fold change relative to the controls with no IGFBP-3 (A) or using 300 μL of the medium not incubated with cells (B and C) was calculated. The curves (A) were fit with a nonlinear regression curve using GraphPad Prism 8.3.1. The data represent the mean ± SD of three separate assays, each of which was performed in triplicate. Asterisks (*) indicate a statistically significant difference from the corresponding cell line control, *p < 0.05 and **p < 0.01, of each cell line. The absence of asterisks indicates no significance (Mann–Whitney test).

Article Snippet: The graphs (E–G) were prepared using the GraphPad 8.3.1 software and summarize the results expressed as the mean ± SD.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, MANN-WHITNEY

Journal: Biochemistry

Article Title: Humanin Blocks the Aggregation of Amyloid- β Induced by Acetylcholinesterase, an Effect Abolished in the Presence of IGFBP-3

doi: 10.1021/acs.biochem.0c00274

Figure Lengend Snippet: The relative abundance of A11-positive prefibrillar oligomers, and to a lesser extent the LOC-positive fibrillar oligomers, is increased in the A549 and H1299 media immunodepleted of HN. Cells (0.2 × 105) were grown in a 10% FBS-supplemented medium for 24 h. The cells were then incubated in a serum-free medium for 72 h, and the medium was collected. The indicated antibodies for the immunodepletion (ID) were added (1:1000 dilution) to the ELISA wells. After blocking the wells, 300 μL of the conditioned medium (0.5 μg/μL, 72 h post serum starvation) was added. Subsequent to incubation overnight, the immunodepleted medium was removed, and the same amount of protein (3 μL of 600 μg/mL total protein) of each sample was spotted onto a nitrocellulose membrane. The blots were either stained with Ponceau (A) or incubated with anti-6E10 (B), anti-A11 (C), or anti-LOC (D) antibodies, and the signal on the membrane was detected using the super signal west pico luminol (chemiluminescence) reagent. The membranes were imaged with a Bio-Rad molecular imager and quantitated with ImageJ software. The dots from five independent assays, each of which was carried out in triplicate, were quantitated, averaged, normalized, and expressed as a fold change relative to the control medium (IGFBP-3). The graphs (E–G) were prepared using the GraphPad 8.3.1 software and summarize the results expressed as the mean ± SD. Asterisks (*) indicate a statistically significant difference from the corresponding cell line control, *p < 0.05 and **p < 0.01, of each cell line. The absence of asterisks indicates no statistical significance (Mann–Whitney test).

Article Snippet: The graphs (E–G) were prepared using the GraphPad 8.3.1 software and summarize the results expressed as the mean ± SD.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Blocking Assay, Staining, Software, MANN-WHITNEY

Journal: Biochemistry

Article Title: Humanin Blocks the Aggregation of Amyloid- β Induced by Acetylcholinesterase, an Effect Abolished in the Presence of IGFBP-3

doi: 10.1021/acs.biochem.0c00274

Figure Lengend Snippet: Exogenously added IGFBP-3 increases the relative abundance of A11-positive prefibrillar oligomers and to a lesser extent the LOC-positive fibrillar oligomers in both A549 and H1299 media. Cells (0.2 × 105) were grown in a 10% FBS-supplemented medium for 24 h. The cells were then incubated in a serum-free medium for 72 h, and the medium was collected. IGFBP-3 was added exogenously to 10 μL of the conditioned medium (0.5 μg/μL, 72 h post serum starvation) and allowed to incubate for 2 h. Following the incubation, 3 μL portions of the samples were spotted onto a nitrocellulose membrane. The blots were stained with either A11 or LOC antibodies, and the signal on the membrane was detected using the super signal west pico luminol (chemiluminescence) reagent. The blots were then imaged with a Bio-Rad molecular imager and quantitated with ImageJ software. The dots from five independent assays, each of which was performed in triplicate, were quantitated, averaged, normalized, and expressed as fold change relative to the control with no added IGFBP-3. GraphPad 8.3.1 software was used in order to prepare the graphs, which summarize the results and are expressed as mean ± SD. Asterisks (*) indicate a statistically significant difference from the corresponding cell line control, *p < 0.05 and **p < 0.01, of each cell line. The absence of asterisks indicates no significance (Mann–Whitney test).

Article Snippet: The graphs (E–G) were prepared using the GraphPad 8.3.1 software and summarize the results expressed as the mean ± SD.

Techniques: Incubation, Staining, Software, MANN-WHITNEY

Journal: Biochemistry

Article Title: Humanin Blocks the Aggregation of Amyloid- β Induced by Acetylcholinesterase, an Effect Abolished in the Presence of IGFBP-3

doi: 10.1021/acs.biochem.0c00274

Figure Lengend Snippet: Immunodepletion of HN from an A549 or H1299 cell-conditioned medium increases the relative amount of oligomer vs the total amount of Aβ, decreasing cell viability and increasing apoptosis. (A and B) Cells (0.2 × 105 cells per well) were seeded in 96-well plates in a 10% FBS-supplemented medium. The next day, the cells were incubated in a serum-free medium for 72 h and then immunodepleted (ID) of AChE, HN, or IGFBP-3 as described in the Experimental Procedures and in the Figure 9 legend. The antibodies 6E10 or 4G8 were bound (1:1000 dilution) to the ELISA wells. The wells were blocked and then incubated with 300 μL of the ID medium (0.5 μg/μL). Biotin-4G8 was then added, and the signal was processed as described in the Experimental Procedures. The fold change relative to the controls using anti-6E10 and anti-4G8 antibodies and 300 μL of the medium not incubated with cells was calculated. Viability (C) or apoptosis (D) of the A549 or H1299 cells was assessed by the MTT assay or the annexin V method, respectively. Cells were seeded in 96-well plates at 0.2 × 105 cells per well in 200 μL of a 10% FBS-supplemented medium. The next day, the cell monolayers were incubated in a serum-free medium for 12 h and then treated with the immunodepleted medium for 48 h, with the medium containing the specific components in the various treatments replaced every 12 h. Data were processed using the GraphPad 8.3.1 software. The graphs summarize the results expressed as the mean ± SD of three separate experiments, each of which was performed in triplicate. Asterisks (*) indicate a statistically significant difference relative to the control. The absence of asterisks indicates no significance (Mann–Whitney test, *p < 0.05 and **p < 0.01).

Article Snippet: The graphs (E–G) were prepared using the GraphPad 8.3.1 software and summarize the results expressed as the mean ± SD.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, MTT Assay, Software, MANN-WHITNEY