Fig. S3.

Establishment of Axl^−/−Tyro3^−/−Gas6^−/− NIH 3T3 (TKO) cells. (A) Gas6-dependent efferocytosis activity of NIH 3T3 cells. Parental NIH 3T3 or TKO cells (0.5 × 10⁵ cells) were incubated at 37 °C for 65 min with 1.0 × 10⁶ pHrodo-labeled apoptotic thymocytes in DMEM containing 5 mg/mL BSA, and 10 nM mProS or mGas6. After incubation, the cells were examined by flow cytometry (Upper), or observed by fluorescence microscopy (Lower). (Scale bar, 20 μm.) (B) Expression of TAM receptors and ligands in NIH 3T3 cells. RNA from NIH 3T3 cells was subjected to real-time RT-PCR for Tyro3, Axl, Mer, ProS, and Gas6. Each mRNA level is expressed relative to the β-actin mRNA level. (C) CRISPR/Cas9-mediated knock out of the Axl, Tyro3, and Gas6 genes in NIH 3T3 cells. The target sequences (Upper) for the Axl, Tyro3, and Gas6 genes and the sequences of the mutated alleles (Lower) in TKO cells are shown. The intron sequence in Gas6 gene is shown in small letters. The protospacer sequence is in light blue, and the protospacer-adjacent motifs (PAM) are in green. The number of added or deleted nucleotides is shown at Right. The mutation caused homozygous (Axl and Tyro3) or heterozygous truncations.