Fig 2: The effect of Flot2 knock-down on the expression of selected genes and flotillin-1 and -2 levels in Raw264 cells. Cells were transfected with Flot2-specific shRNA variants No. 1, 3, 5 (Flot2 shRNA) or control shRNA (NC1, NC2 shRNA). RT-qPCR analysis of Flot2 (A), Flot1 (E), Cd14 (F) and Tlr4 (G) transcripts quantified as in Fig. 1. B–D The abundance of flotillin-1 and -2 determined by immunoblotting (B) followed by densitometric analysis (C, D). The flotillins were quantified in indicated cells relative to actin. Positions of molecular weight markers are shown on the right in kDa. Jak1 and actin were visualized to verify equal loading of protein between wells. Data shown are mean ± SD from at least three experiments. Significantly different values as indicated by 1-way ANOVA with Scheffe's post hoc test are marked. In (A, C, D) the p values for individual Flot2 shRNA variants were < 0.001 relative to both NC variants

Fig 3: GABA antagonizes the pro-proliferative effects of BCAT1 in NPC. (A) CCK-8 assay indicating the effects of GABA at different concentration on 5-8F and 6-10B cells. (B, C) plate clone formation and EdU assays showing the inhibitory effects of GABA at 20mM on proliferation of 5-8F cells. (D) Western blot demonstrating the levels of BCAT1 and FLOT2 in 5-8F cells treated by GABA at 20mM. (E–G) CCK-8, plate clone formation, and EdU assays showing inhibitory effects on 6-10B-FLOT2 cells treated by GABA at 20mM. **, P < 0.01, ***; P < 0.001.

Fig 4: FLOT2 promotes proliferation via positively regulating BCAT1 in NPC. (A) Western blot assay indicating the level of FLOT2 and BCAT1 in 5-8F-shFLOT2, 5-8F-shFLOT2+BCAT1, 6-10B-FLOT2, 6-10B-FLOT2+shBCAT1, and control cells. (B–D) CCK-8, plate clone formation, and EdU assays showing the growth and proliferation abilities of 5-8F-shFLOT2, 5-8F-shFLOT2+BCAT1, 6-10B-FLOT2, 6-10B-FLOT2+shBCAT1, and control cells in vitro. 5-8F-shFLOT2, 5-8F-shFLOT2+BCAT1, 6-10B-FLOT2, 6-10B-FLOT2+shBCAT1, and control cells were subcutaneously implanted into nude mice (n = 5 each group) for xenografts formation to analyze the proliferation in vivo. The pictures (E), the volumes (F), and weights (G) of xenografts were presented. *, P <0.05, **, P < 0.01, ***; P < 0.001.

Fig 5: FLOT2 regulates BCAT1 and promotes cell proliferation via maintaining c-Myc in NPC. (A) Western blot and qPCR assays indicating the level of c-Myc mRNA and protein in 5-8F-shFLOT2, 6-10B-BCAT1 and control cells. (B) Western blot showing the level of c-Myc, BCAT1 and FLOT2 in 5-8F-shFLOT2, 5-8F-shFLOT2+c-Myc, 5-8F-shFLOT2+c-Myc+siBCAT1, 6-10B-BCAT1, 6-10B-FLOT2+sic-Myc, 6-10B-FLOT2+sic-Myc+BCAT1, and control cells. (C–E) CCK-8, plate clone formation, and EdU assays showing the growth and proliferation abilities of 5-8F-shFLOT2, 5-8F-shFLOT2+c-Myc, 5-8F-shFLOT2+c-Myc+siBCAT1, 6-10B-BCAT1, 6-10B-FLOT2+sic-Myc, 6-10B-FLOT2+sic-Myc+BCAT1, and control cells. *, P <0.05, **, P < 0.01, ***; P < 0.001.