Blasticidin s

Lenti-X 293T cells (Takara) were cotransfected with pCSII plasmids, pCMV-VSV-G-RSV-Rev (RIKEN BRC #RDB04393), and pCAG-HIVgp (RIKEN BRC #RDB04394). The medium containing the lentiviruses was collected and filtered through a 0.45 μm pore-size membrane filter 72 h after transfection. hTERT-RPE1 cells were combined with a 1:10 lentivirus solution and the cells selected using 20 μg/ml blasticidin S (FUJIFILM Wako).

Dictyostelium discoideum strains used are listed in Supplementary Table 1. The pzoA⁻ strain was constructed using the vector pKOSG-IBA-dicty1 (iba) following the manufacturer’s instructions^{57 (link)}. The 5′- and 3′-flanking sequences were generated using polymerase chain reaction (PCR) and cloned into pKOSG-IBA-dicty1 (Supplementary Fig. 7a). Primer pairs used for PCR were pzA_KO_LA1/pzA_KO_LA2 (5′-) and pzA_KO_RA1/pzA_KO_RA2 (3′-) (Supplementary Table 2). The pzoA gene disruption was confirmed via PCR. Cells were grown axenically in HL5 medium (Formedium, UK) in culture dishes at 21 °C. Transformants were maintained in 20 μg mL⁻¹ G418 or 10 μg mL⁻¹ blasticidinS (both from Fujifilm Wako, Japan).

Axenic strain Ax2 was cultured and transformed as described elsewhere [4 (link), 23 (link), 26 (link)]. For knockin transformation, cells were washed and suspended with ice-cold EP buffer (6.6 mM KH₂PO₄, 2.8 mM Na₂HPO₄, 50 mM sucrose, pH 6.4) to 1 × 10⁷ cells/ml. 800 μl of cell suspension mixed with 10–40 μg of NotI digested knockin vector in a 4-mm width cuvette was subjected to electroporation (5 s separated two pluses with 1.0 kV and 1.0 ms of the time constant) by using a MicroPulser (Bio-Rad). These cells were plated on 4–6 pieces of 10 cm-petri dishes with HL5 medium and incubated at 22 °C for 18 h under the non-selective condition, then were cultured in the presence of 12.5 μg/ml Blasticidin S (Wako). After 4–7 days, colonies of candidate recombinants were manually picked and were transferred into 96-well plates, then subjected for the genome check by PCR and Southern blotting analysis. Cre-recombinase mediated removal of the selection cassette was performed as described previously [23 (link)]. Briefly, NLS-Cre was transiently expressed by introducing pDEX_NLS-Cre. Candidate clones were selected in the presence of 5 μg/ml of G418 for 2–5 days followed by an additional few days’ culture in the absence of G418 (LifeTechnologies). Optionally, single cell sorting of isolated clones was performed by a JSAN cell sorter equipped with a CloneMate module (Bay Bioscience).

C3H10T1/2 culture, retrovirus transfection, and establishment of Venus- (Nagai et al., 2002 (link)) or Venus-Mkx C3H10T1/2 cells were performed as previously described (Nakamichi et al., 2016 (link)). Briefly, C3H10T1/2 cells were cultured in alpha minimal essential medium (MEMα) with 10 v/v% fetal bovine serum (FBS) and 1 v/v% penicillin/streptomycin (15140-122, Gibco, MA, United States). Retrovirus infection was performed in the supernatant of retrovirus vector (pMIGR/Venus, pMIGR/Venus-Mkx) transfected PLAT-E cells. In order to clearly observe the cell shape, we simultaneously introduced mCherry in both cell lines using the supernatant of pMIGR/mCherry-transfected PLAT-E cells. Stable cell lines were established by 1 μg/mL puromycin (ant-pr-1, InvivoGen, CA, United States) and 10 μg/mL blasticidin S (026-18711, FUJIFILM Wako Pure Chemical Corp., Osaka, JAPAN) selection for 1 week.