To study the role of γ-secretase activating protein (GSAP) in regulating APP processing and to study the reproducibility of GSAPs role in specific γ-secretase cleavage of APP.
With all the published data on this subjectit is clear that GSAP is not a specific activator of γ- secretase cleavage of APP and further characterization of GSAP function is needed. However, it will be interesting to pursue how GSAP silencing reduces APP levels. One possibility is that APP transcript levels are reduced or APP protein degradation is enhanced.
siRNAs were purchased from Invitrogen (stealth siRNA). The sequences and the gene s are supplied in Supplementary Table 1.
siRNA transfection for HeLa-swAPP cells
Cells were transfected with a final concentration of 5 nM using Oligofectamine (Invitrogen) as a transfection reagent at a concentration of 0.3 m in a total volume of 100 m following the manufacturer’s instructions. Each siRNA transfection was performed in quadruplicate. After 24 h the transfection mix was replaced with fresh culture medium. 69 h after transfection, medium was again replaced with 100 m fresh medium containing 10% Alamar lue™ (AbD Serotec). Supernatants were collected and assayed for Aβ and sAPPβ, as described below. The cells were lysed with 50 m lysis buffer (1% NP-40, 0.1% SDS and protease inhibitor cocktail tablet) for 20 min on ice.
Alamar Blue assay
For cell viability measurements using Alamar Blue, the medium of transfected cells (siRNA or plasmid) was replaced with normal medium containing 10% Alamar lue. The final volume in each well was 100 m. 3 h after the medium change, cell viability was monitored using Fluoroscan Ascent Cf (Labsystems), with excitation wavelength 544 nm and emission at 590 nm.
Electrochemiluminescence (ECL) detection of Aβ, sAPPβ
An electrochemiluminescence assay (Meso Scale Discovery, MD) was performed to determine the amount of secreted Aβ40, sAPPα and sAPPβ in the cell culture medium 10. For the measurement of Aβ38, 40 and 42, triplex plates were used from conditioned supernatants collected for 12 h. Pre-coated plates were blocked with Tris Buffered Saline containing Tween, containing 3% Blocker A, for 1 h at room temperature on a shaker at 750 rpm. After washing, 10 m of the cell culture supernatant was added to each well along with 10 m of detection antibody followed by incubation for 2 h at room temperature on a shaker at 750 rpm. After washing detection was performed in 35 m 2X MSDT read buffer and read with the Sector Imager 6000.
Western lotting: After 72 h of siRNA transfection, cells were lysed in buffer containing 1% NP-40 and 0.1% SDS and protease inhibitors (Roche). Equal amounts of the lysate (according to the protein content quantified by BCA assay (Pierce)) were run on 4-12% BIS-TRIS gels (Invitrogen). The gel was blotted onto a nitrocellulose membrane (BioRad) and probed with the respective antibodies: anti-APP, C-terminal antibody: SIGMA (F3165-1MG); 6E10 anti-Aβ recognizing APP antibody Covance; anti-Nicastrin antibody (2332-1) Epitomics; GAPDH antibody: Meridian Science.
Immunofluorescence: Cells were reverse transfected either with MEDGC or siRNAs against APP or GSAP on chambered coverslides and 72 h later fixed, permeabili ed, blocked and immunostained with 6E10 anti-APP antibody. The signal was visualised by the use of Alexa488 or Alexa546coupled anti-mouse secondary antibody. Nuclei were visualised by DAPI staining.
Cells were reverse transfected either with MEDGC or siRNAs against GSAP and 69 h later, cells were treated with DMSO or Imatinib (10 μM, Enzo Life Sciences) for 3 h. Treatment was done with culture medium containing 10% Alamar Blue. The final volume of culture medium in each well was 100 μ . After 72 h, cell viability was assessed by Alamar Blue assay, supernatants were collected and assayed for Aβ and sAPPβ.
Cell cytotoxicity assay
The cell cytotoxicity assay was carried out using the Cytotoxicity Detection Kit (Roche) by measuring the activity of released lactate dehydrogenase into the culture medium. The culture supernatant was collected from the cells (with and without Triton X-100 treatment). Samples were further diluted and processed according to manufacturers protocol. The increase in amount of enzyme activity in the supernatant directly correlates the amount of formazan formed, which is proportional to the number of lysed cells. Absorbance was measured at 492 nm with reference wavelength at 620 nm on a plate reader spectrophotometer.
Total RNA from cells was isolated using TRI Reagent® (Sigma-Aldrich) following manufacturer’s protocol. 1 μg of total RNA was used for reverse transcription with iScript™ cDNA synthesis kit (Bio-Rad) according to the manufacturer’s protocol. Real-time PCR was performed using iTaq™ Universal SYBR® Green supermix (Bio-Rad) following manufacturer’s instructions. Relative gene expression levels were calculated with the ΔΔCt method using GAPDH for normalization.
L.R acknowledges the financial support from the Velux Foundation, the Swiss National Science Foundation grant, the Bangerter Stiftung, the Baugarten Stiftung and the Synapsis Foundation. L.R and V. U acknowledge the funding support from the European Neuroscience Campus of the Erasmus Mundus Program.
We thank G. Yu for the HeLa-swAPP cells.
All the experiments were conducted according to the standard ethical guidelines.
No fraudulence is committed in performing these experiments or during processing of the data. We understand that in the case of fraudulence, the study can be retracted by Matters.
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