Fig 1: Ectopic expression of SLAMF7 variant X secreted sSLAMF7 and enhanced multiple myeloma cell growth. (A) Structure-based prediction of SLAMF7 variant 1-variant X interactions using the AlphaFold2 program. (B) We used the lentiviral vector CSII-CMV-MCS-IRES-VENUS (provided by Dr. Hiroyuki Miyoshi, RIKEN BioResource Center, Ibaraki, Japan) containing the coding regions of SLAMF7 variant 1 and SLAMF7 variant X cDNA for gain-of-function experiments. Whole cell lysates and supernatants were prepared from RPMI8226 cells transduced with mock, SLAMF7 variant 1, or SLAMF7 variant X after 24 hours (hr) of culture. Immunoblotting was carried out according to a standard method using the following antibodies: anti-SLAMF7 (#98611), anti-BSA (#23053) (Cell Signaling Technology, Beverly, MA, USA); and anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The immunoblot shows the expression levels of SLAMF7 protein, with GAPDH as the loading control of cell lysate, and BSA as the loading control of supernatant (left panel). The sSLAMF7 levels in the culture supernatants were measured using ELISA (right panel). Bars indicate the mean of 3 independent experiments. P value was determined by one-way ANOVA with Tukey’s multiple comparison test. (C) SLAMF7-negative (SLAMF7–) RPMI8226 and KMM.1 cells and SLAMF7-positive (SLAMF7+) KMS28-BM and MM.1S cells were cultured with the supernatant derived from 1x105 RPMI8226 cells transduced with SLAMF7 variant 1 or SLAMF7 variant X after 24 hr of culture. Cell proliferation was assessed after 72 hr using the MTT reduction assay and is shown relative to the variant 1 group. Bars indicate the means of 3 independent experiments. P value was calculated using a paired Student t test.