Pei prime

HEK293T cells were
seeded in 24-well plates at the density of 2×105 cells/well.
Twenty-four hours later, the cells were cotransfected with plasmids
encoding HA-Cyclin D1 (0.5 μg) with Flag-DYRK1A or FLAG-GSK3β
or an empty vector (0.2 μg) using PEI Prime (Sigma-Aldrich).
Forty-eight hours later, the cells were treated with inhibitors CX-4945
(10 μM), harmine (10 μM), 1-azakenpaullone (10 μM),
or DMSO for 3 h and lysed in RIPA buffer containing protease inhibitors
(Sigma-Aldrich) and phosphatase inhibitors (Calbiochem). Total cell
proteins (10 μg) were separated by 12% SDS-PAGE and transferred
to the PVDF membrane (ThermoScientific). Proteins were analyzed by
Western Blot using the anti-FLAG monoclonal antibody (Sigma-Aldrich;
F3165) for DYRK1A and GSK3β detection, the anti-HA monoclonal
antibody (Cell Signaling; C29F4) for Cyclin D1, and anti-phospho-Cyclin
D1 (Thr286) (Cell Signaling; D29B3) for phospho-Cyclin D1 relevant
HRP-conjugated secondary antibodies.

HEK293T cells were grown on
a μ-Slide 8 well (IBIDI) to 50%–70% confluency. The plasmids
expressing the desired proteins, mCherry-DYRK1A or mCherry-GSK3β
and eGFP-NFATc1, were transiently cotransfected for 24 h with PEI
Prime (Sigma-Aldrich). Cells were pretreated with inhibitors CX-4945
(5 μM), harmine (5 μM), or DMSO for 3 h and then stimulated
with ionomycin (Thermo Fisher Scientific) for 1 h. Cells were washed
with 1 mL of PBS, and the nuclei were stained with Hoechst 33258 (ThermoScientific)
for 10 min at 37 °C and fixed with 4% paraformaldehyde in phosphate-buffered
saline (PBS) for 10 min at 25 °C. Images were collected with
a Zeiss Axio Observer 3 fluorescence microscope and analyzed in ZEN
Blue edition software.

Based on the procedure detailed in Grygier et al.^{34 (link)}, the experiment was conducted as follows: HEK293T cells were seeded in 24-well plates at the density of 2 × 10⁵ cells/well. After 24 h the cells were co-transfected with plasmids encoding HA-CyclinD1 (0.5 µg) with Flag-DYRK1A or empty vector (0.2 µg) using PEI Prime (Sigma-Aldrich) transfection reagent. 48 h later cells were treated with compounds (10 μM) or DMSO for 6 h and lysed in RIPA buffer supplemented with protease (Sigma-Aldrich) and phosphatase (Calbiochem) inhibitors. Total cell proteins (10 µg, quantified by the BCA method) were separated by 12% SDS-PAGE and transferred to the PVDF membrane (ThermoScientific). Proteins were analysed by Western Blot using anti-FLAG monoclonal antibody (Sigma-Aldrich; F3165) for DYRK1A detection, anti-HA monoclonal antibody (Cell Signaling; C29F4) for CyclinD1 and anti-phoshoCyclinD1 (Thr286) (Cell Signaling; D29B3) for phosphoCyclinD1 and relevant HRP-conjugated secondary antibodies.