Peanut lectin solution

C. elegans embryonic cells were generated as previously described (Strange et al., 2007 (link)). Worms were grown on 10-cm enriched peptone plates with NA22 E. coli. NA22 bacteria grow in very thick layers that provide an abundant food source for large quantities of worms. Synchronized gravid hermaphrodites were bleached to release eggs and washed with sterile egg buffer (118 mM NaCl, 48 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, and 25 mM HEPES, pH 7.3, 340 mOsm, adjusted with sucrose). The isolated eggs were separated from debris by centrifugation in a 30% sucrose solution. Chitinase (1 U/ml; Sigma-Aldrich) digestion was performed to remove eggshells. The embryo cells were dissociated by pipetting and filtered through a sterile 5-µm Durapore filter (Millipore). The cells were plated on glass coverslips coated with a peanut lectin solution (0.5 mg/ml; Sigma-Aldrich) and cultured in L15 medium (Gibco) supplemented with 50 U/ml penicillin, 50 µg/ml streptomycin, and 10% FBS (Invitrogen) for 72–96 h.

To express pezo-1 in Sf9 cells, we produced recombinant baculoviruses, according to the manufacturer’s instructions (Bac-to-Bac expression system; Invitrogen). To generate this baculovirus, we used a pFastBac construct (Epoch Life Science) containing an 8× histidine–maltose binding protein tag and a synthesized pezo-1 isoform G nucleotide sequence (one of the longest isoforms according to RNA sequencing; wormbase.org release WS280). For expression of pezo-1 R2373K, the construct contained an 8× histidine–maltose binding protein tag and a synthesized pezo-1 isoform G with the R2373K point mutation. We infected Sf9 cells with either pezo-1 baculovirus for 48 h. Infected cells were plated on glass coverslips coated with a peanut lectin solution (1 mg/ml; Sigma-Aldrich) for patch-clamp experiments.