Hela-sweAPP are cultured and used as described.
siRNAs were purchased from Invitrogen (stealth siRNA). The sequences and the gene IDs 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 mL in a total volume of 100 mL 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 mL fresh medium containing 10% Alamar Blue™ (AbD Serotec). Supernatants were collected and assayed for Aβ and sAPPβ, as described below. The cells were lysed with 50 mL 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 Blue. The final volume in each well was 100 mL. 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, and sAPPα
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 mL of the cell culture supernatant was added to each well along with 10 mL 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 mL 2X MSDT read buffer and read with the Sector Imager 6000.
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.
Cells were reverse-transfected either with MEDGC or siRNAs against APP or GSAP on chambered coverslides and 72 h later fixed, permeabilised, blocked and immunostained with 6E10 anti-APP antibody. The signal was visualised by the use of Alexa488 or Alexa546-coupled 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 μL. 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 with 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.