Fig 2: The expression of BECN1 affects the migratory ability of NSCLC cells. (a) H1299 cells were transfected with BECN1 siRNAs and wound healing assay was performed. The photographs were taken at 0 and 12 h (Left panels). Cell migration was quantified by measuring the difference in area between the leading edge and the initiation edge of the experiment. The wound area was assessed by the ImageJ software. **, P≤ 0.01 (Right panel). (b) BECN1 was knocked down in H1299 cells by specific siRNAs and transwell assay was performed. After 18 h, the migrated cells were fixed and stained with 2% crystal violet. The photographs were taken under 100× magnification (Left panels). The dyes were dissolved by 10% acetic acid and the quantification of cell migration was determined by the absorbance at 595nm. Data represent the average of three independent experiments. ***, P≤ 0.001 (Right panel).

Fig 3: BECN1 knockdown does not significantly affect the proliferation of NSCLC cells. (a) H1299 cells were transfected with siRNAs specific for BECN1. Twenty-four hours later, cell growth assay was performed. At the indicated times, cells were fixed with 4% formaldehyde and then stained with 2% crystal violet. The dyes were finally dissolved by 10% acetic acid and the relative proliferation was determined by the absorbance at 595nm. Data represent the average of three independent experiments. ns, P >0.05 (Left panel). The knockdown efficiency of the siRNAs targeting BECN1 was determined by Western blot using anti-BECN1 antibody (Right panel). (b) H1299 cells were transfected with BECN1 siRNAs for 24 h and colony formation assay was performed. After ten days, the cells were fixed with 4% formaldehyde and then stained with 2% crystal violet. The colonies were photographed (Left panel). The dyes were dissolved by 10% acetic acid and the quantification of colony formation was determined by the absorbance at 595nm. Data represent the average of three independent experiments. ns, P >0.05 (Right panel). (c) A549 cells were transfected with BECN1 siRNAs. Twenty-four hours later, cell growth assay was performed. At the indicated times, cells were fixed with 4% formaldehyde and then stained with 2% crystal violet. The dyes were finally dissolved by 10% acetic acid and the relative proliferation was determined by the absorbance at 595nm. Data represent the average of three independent experiments. ns, P >0.05 (Left panel). The knockdown efficiency of the siRNAs targeting BECN1 was determined by Western blot using anti-BECN1 antibody (Right panel). (d) A549 cells were transfected with BECN1 siRNAs for 24 h and colony formation assay was performed. After ten days, the cells were fixed with 4% formaldehyde and then stained with 2% crystal violet. The colonies were photographed (Left panel). The dyes were dissolved by 10% acetic acid and the quantification of colony formation was determined by the absorbance at 595nm. Data represent the average of three independent experiments. ns, P >0.05 (Right panel).

Fig 4: H1299 cells stably knocking down BECN1 show reduced migratory ability. (a) H1299 cells stably knocking down BECN1 were used to perform soft agar assay. After two weeks, the Photographs were taken under 40× magnification. Scale bar: 200μm (Left panel). Colonies larger than 50μM were scored. Data represent the average of three independent experiments (mean±SD). ns, P >0.05 (Right panel). (b) Transwell assay was performed using H1299 cells stably knocking down BECN1. The migrated cells were fixed and stained with 2% crystal violet. The photographs were taken under 100× magnification. (Left panels). The dyes were dissolved by 10% acetic acid and the quantification of cell migration was determined by the absorbance at 595nm. Data represent the average of three independent experiments. **, P≤ 0.01 (Right panel). (c) H1299 stable cell line with BECN1 knockdown were stained with the fluorescent Phalloidin and DAPI. Photographs were taken under 200× magnification using fluorescence microscope. The white arrow indicated filopodium. Scale bar: 50μm.

Fig 5: The de-ubiquitination of Vimentin by USP14 depends on the expression of BECN1. (a) H1299 cells were transfected with pCMV-HA-USP14 plasmid. Forty-eight hours later, HA-USP14 was immunoprecipitated using rabbit anti-HA antibody followed by Western blot. BECN1 was detected using mouse anti-BECN1 antibody and HA-USP14 was detected using mouse anti-HA antibody. (b) H1299 cells were transfected with pCMV-HA-BECN1 plasmid. Forty-eight hours later, HA-BECN1 was immunoprecipitated using mouse anti-HA antibody followed by Western blot. USP14 was detected using rabbit anti-USP14 antibody and HA-BECN1 was detected using rabbit anti-HA antibody. (c) H1299 cells were transfected with pCMV-HA-BECN1 plasmid. Forty-eight hours later, immunoprecipitation was performed using normal rabbit IgG and rabbit anti-USP14 antibody followed by Western blot. USP14 was detected using mouse anti-USP14 antibody and Vimentin was detected using mouse anti-Vimentin antibody. (d) H1299 cells were transfected with BECN1-specific siRNA. Forty-eight hours later, immunoprecipitation was performed using normal rabbit IgG and rabbit anti-USP14 antibody followed by Western blot. USP14 was detected using mouse anti-USP14 antibody and Vimentin was detected using mouse anti-Vimentin antibody. (e) H1299 cells were transfected with pCMV-HA-USP14 alone or co-transfected with pCMV-HA-USP14 and pCMV-HA-BECN1. Forty-eight hours later, immunoprecipitation was performed using mouse anti-Vimentin antibody. The K48-linked ubiquitination was detected using K48-linkage specific polyubiquitin antibody. Vimentin was detected using rabbit anti-Vimentin antibody. (f) H1299 cells were transfected with pCMV-HA-USP14 alone or co-transfected with pCMV-HA-USP14 and BECN1 siRNA. Forty-eight hours later, immunoprecipitation was performed using mouse anti-Vimentin antibody. The K48-linked ubiquitination was detected using K48-linkage specific polyubiquitin antibody. Vimentin was detected using rabbit anti-Vimentin antibody.