Turbofect

For co-IP, CHME3 cells (3 × 10⁶) were transfected with 2 μg of APP-Flag or HA-tagged forms of HIV-1 Gag alone, or in combination using 15 μl Turbofect (Thermo scientific). Total DNA amount in each group was kept constant with addition of empty vector pcDNA3.1⁻. Soluble cell extracts were prepared 48 h post-transfection as described^{20 (link)} and precleared with protein G-sepharose. A concentration of 27 μl of the input samples was taken and the remainder of the cell extract was incubated with 2 μl of the mouse anti-APP antibody (Invitrogen, 130200) and protein G-sepharose for 1 h at 4 °C. Immune complexes were then washed and boiled with Laemmli buffer and subjected to WB analysis. For co-IP of endogenous APP with Gag, 1 × 10⁶ CHME3 4 × 4 cells were infected with WT HIV-1 or mock virus 48 h before cell lysates were subjected to co-IP as described above. For GBP-binding assay, 293T cells (3 × 10⁶) were transfected with 2 μg of a GFP-expressing control vector (GFP) or N′-GFP-APP₇₇₀ (GFP-APP) along with 2 μg of HA-tagged forms of HIV-1 Gag (Gag-HA, Gag-N-Myr-HA or Gag-MA: -20-HA, -40-HA, -60-HA, and -80-HA) using 15 μl Turbofect (Thermo scientific). Two days post-transfection, cells were lysed with 1 ml cold NP-40 lysis buffer, subjected to GBP-binding assay followed by WB analysis as described in ref. ^{20 (link)}.

SH-SY5Y cells were cultured
in high-glucose Dulbecco’s modified Eagle’s medium (DMEM,
Gibco, 11965–084) supplemented with 1% penicillin/streptomycin
(Gibco, 15140–122) and 10% (v/v) fetal bovine serum (FBS, Corning,
35–010-CV). The cells were maintained in 10 cm culture dishes
(α-plus, 16203–1SS) at 37 °C in a saturated humidity
atmosphere containing 95% air and 5% CO₂. For assays measuring
cell viability, ROS generation, mitochondrial function verification,
caspase 1/3 activity, and autophagy activity, SH-SY5Y cells were transfected
with a specific amount WT α-Syn for 24 h using TurboFect (Thermo
Fisher, R0531), following the vendor’s instructions, either
in the absence or presence of CAE. For FCS experiments, SH-SY5Y cells
were plated at a density of 4.5 × 10⁵ cells/plate
and transfected with eGFP-tagged WT α-Syn/mutant variants and
mApple-tagged WT α-Syn/mutant variants for 24/48 h using TurboFect
(Thermo Fisher, R0531), as per the vendor’s instructions, in
the absence or presence of CAE. Before confocal imaging and FCS measurements,
the medium was replaced with phenol red-free DMEM (Gibco, 31053–028).
During data acquisition, cells were maintained at 37 °C in a
saturated humidity atmosphere containing 95% air and 5% CO₂ in a culture dish microincubator (Warner, DH-40iL).

Cell lines MCF7, T47D and ZR75-1 were seeded in 6 well plates on glass slides and transfected the following day with 4 μg of EYFP or RASSF10-EYFP using Turbofect (Thermo Fisher Scientific). 48 h or 72 h after transfection cells were fixed with formaldehyde, stained with DAPI (0.1 μg/mL in PBS, Sigma Aldrich, Taufkirchen, Germany) and embedded in Mowiol (Sigma Aldrich). Transfected cells (n > 100) were analysed for normal, condensed, fragmented or missing nucleus using Axio Observer Z1 (Zeiss, Jena, Germany). Volocity Software (Perkin Elmer, Baesweiler, Germany) was used for nuclei shrinkage measurements. For cell cycle/apoptosis analysis cells were grown on 10 cm plates and were transfected with 10 μg of EYFP or RASSF10-EYFP using Turbofect (Thermo Fisher Scientific). After 48 h and 72 h cells were isolated using Trypsin (Gibco, Thermo Fisher Scientific). Cells were fixed with ethanol over night at −20 °C. The following day cells were treated with 50 μg/mL RNase A for 30 min at 37 °C. For flow cytometry assay cells were stained with 50 μg/mL propidium iodide prior to measurement of DNA content in FACSCantoII (BD Biosciences, Heidelberg, Germany). FACSDiva Software (BD Biosciences) was used for measurement and gating to distinguish transfected fluorescence cells and to determine sub G1, G0-G1, S and G2-M phase of the cell cycle.

Low molecular weight poly(I:C) and Escherichia coli LPS were purchased from Invivogen and Sigma, respectively. For induction with poly(I:C), HEK-293TO cells were plated at 2.5 × 10⁵ cells/mL in 24-well plates and transfected with 1 µg/mL poly(I:C) using TurboFect (Thermo Scientific); 2 µL of TurboFect was used per µg of poly(I:C). After 24 h, cells were washed with PBS and harvested for RNA using the MagMAX-96 RNA Isolation kit (Thermo Scientific). THP-1 cells were differentiated into macrophage-like cells with 5 ng/mL of PMA (Sigma) for 72 h. Cells were washed with PBS and induced with media containing LPS (1 ng/mL). Cycloheximide was purchased from Sigma, and 2.5 × 10⁵ HEK-293TO cells were treated with 50 µg/mL Cycloheximide for 4 h. Cells were washed with PBS and harvested for RNA as above. Differentiated Mouse BMDMs were treated with LPS (100 ng/mL) or poly(I:C) (5 µg/mL) for immunostimulation.

HEK-293 cells were used for transfection experiments. Cells were routinely screened for mycoplast contaminations. Cells were transfected with empty pcDNA3 as a control or pcDNA3 containing the cDNA for human WT, Cys42Ser, Cys117Ser, Cys195Ser, Cys117/195Ser or Cys42/117/195Ser PKGIα (2 µg DNA/well) using Turbofect (Turbofect, Thermo Scientific, Venlo, Limburg, The Netherlands). After 24 hours, cells were treated with NCA (100 µmol/L, 30 min), AS (500 µmol/L, 15 min) or vehicle (1% DMSO or 100 µmol/L decomposed NCA for NCA; 10 mmol/L NaOH, 50 µmol/L nitrite (NO₂⁻), 500 µmol/L decomposed AS for AS). Western immunoblot analysis was performed to assess oxidative PKGIα modifications or in vitro kinase assays to assess PKGIα activity.

HEK293T cells were collected by trypsin digestion and plated on 10 cm cell culture dishes at a density of 1–2 × 10⁷ cells/plate with DMEM complete medium. The total area occupied after cell attachment was 80–90% of the dish area. Transient transfection was initiated after complete cell attachment by incubation in a 37 °C incubator containing 5% CO₂ for 8–24 h according to cell attachment. TurboFect-DNA Mix was prepared according to TurboFect (R0531, Thermo, GER) instructions. Briefly, 1 µg/plasmid and 2 µL TurboFect were added to 200 µL Opti-Medium, gently mixed, and incubated at room temperature for 15 min. Next, TurboFect-DNA Mix was added to the culture dish and the complete medium was replaced after 12 h. The culture was incubated for 48 h. The cells were observed under a microscope, and the medium was collected for the next step.