Fig. 6

In vitro and in vivo effects of knocking out the SNX5 gene in A549 cells. a–e CRISPR–Cas9 mediated SNX5 knockout inhibits macropinocytosis, ATP internalization and suppresses migration/invasion. a Western blot confirmation for SNX5 protein knockout in A549 cells. b A direct count of viable cells of A549 control and A549snx5ko for determination of their respective cell proliferation rates. c Intracellular ATP levels of A549 (control) or A549snx5ko cells incubated with or without ATP, and the intracellular ATP levels were measured by ATP assay. d Fluorescence microscopy of A549 (control) and A549snx5ko cells treated with a known macropinocytosis tracer, TMR-dextran and quantification of intracellular TMR-dextran. e Transwell invasion assay results indicating the ATP-induced cell invasion ability of A549 and A549snx5ko cells, and the quantification of invasive cells. f–h A549 (control) or A549snx5ko cells were subcutaneously injected into flanks of male nude mice (n = 9 per group) to generate tumors. 4 weeks after inoculation, tumors were removed for examination. f Comparison of sizes of surgically removed tumors. g Tumor weights measured after surgical tumor removal. h Weekly tumor growth curves after tumor cell inoculation. i HE staining of A549 and A549snx5ko tumor sections. Experiments were performed in triplicate and data are presented as the mean ± standard deviation. *P < 0.05, **P < 0.01 and ***P < 0.001