P100 dish

For biochemical experiments, HEK293 cells were individually transiently transfected with the following plasmids: 240 ng of HA-TRPM4 WT or HA-TRPM4 N992Q, 2 μg of either TRPM5 WT or TRPM5 N932Q in a P100 dish (BD Falcon, Durham, North Carolina, USA), mixed with 30 μ l of JetPEI (Polyplus transfection, Illkirch, France) and 250 μ l of 150 mM NaCl. The cells were incubated for 48 h at 37°C with 5% CO₂. For electrophysiological studies, T25 cm² flasks of HEK293 cells were transiently co-transfected using X-tremeGENE 9 DNA transfection mix reagent (Roche Diagnostics, IN, USA) with 0.3 μg of WT, mutant TRPM4, or TRPM5 channels. These transfections included 0.2 μg of cDNA encoding CD8 antigen as a reporter gene. Anti-CD8 beads(Dynal, Oslo, Norway) were used to identify transfected cells, and only CD8-displaying cells were analyzed.Cells were used 48 h after transfection.

Human embryonic kidney (HEK293) cells were cultured with Dulbecco's modified Eagle's culture medium supplemented with 4 mM Glutamine, 10% FBS and a cocktail of streptomycin-penicillin antibiotics. For the electrophysiological studies, the cells were transiently transfected with 240 ng of HA-TRPM4 WT or HA-TRPM4 p.A101T, HA-TRPM4 p.Q854R, HA-TRPM4 p.S1044C, HA-TRPM4 p.P1204L, and double variant HA-TRPM4 p.A101T/P1204L in a 35 mm dish (BD Falcon, Durham, North Carolina, USA) mixed with 4 μL of JetPEI (Polyplus transfection, Illkirch, France) and 46 μL of 150 mM NaCl. The cells were incubated for 24 h at 37°C with 5% CO₂. All transfections included 100 ng of eGFP as a reporter gene. Expression of GFP was used to identify transfected cells, for patch clamp experiments.
For the biochemical experiments, HEK293 cells were transiently transfected with 240 ng of either HA-TRPM4 WT, or HA-TRPM4 p.A101T, HA-TRPM4 p.Q854R, HA-TRPM4 p.S1044C, HA-TRPM4 p.P1204L, and double variant HA-TRPM4 p.A101T/P1204L, or empty vector (pcDNA4TO) in a P100 dish (BD Falcon, Durham, North Carolina, USA) mixed with 30 μL of JetPEI (Polyplus transfection, Illkirch, France) and 250 μL of 150 mM NaCl. The cells were incubated for 48 h at 37°C with 5% CO₂.