Fig 2: 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 3: 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.

Fig 4: The cell cycle progression is not affected by BECN1 expression. (a) H1299 cells were transfected with pCMV-HA or pCMV-HA-BECN1. Forty-eight hours later, cells were harvested and analyzed by flow cytometry (Left panel). The quantification of cell number in each phase of the cell cycle derived from left panel was marked with different colors [red: quiescent phase and the first gap phase (G0/G1 phase), green: DNA synthesis phase (S phase), blue: the second gap phase and mitotic phase (G2/M phase)] (Right panel). (b) The mRNA levels of the cell cycle-related genes were determined by Q-PCR in H1299 cells. Data represent the average of three independent experiments (mean±SD). ***, P= 0.001; ns, P >0.05. (c) The protein levels of the cell cycle-related genes were determined by Western blot using indicated antibodies in H1299 cells transfected with BECN1 siRNAs.

Fig 5: BECN1 affects the ubiquitination and expression of Vimentin. (a) H1299 cells were transfected with pCMV-HA-BECN1 plasmid. Forty-eight hours later, Western blot was performed to examine the expression of indicated proteins. (b) H1299 cells were transfected with pCMV-HA-BECN1 plasmid. Forty-eight hours later, total RNA were extracted. The mRNA levels of related genes were determined by Q-PCR. Data represent the average of three independent experiments (mean±SD). ***, P= 0.001; ns, P >0.05. (c) H1299 cells were transfected with BECN1 siRNAs. Forty-eight hours later, the indicated proteins were detected by Western blot. (d) H1299 cells were transfected with BECN1 siRNAs. Forty-eight hours later, total RNA were extracted. The mRNA levels of related genes were determined by Q-PCR. Data represent the average of three independent experiments (mean±SD). ***, P= 0.001; ns, P >0.05. (e) 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. Vimentin was detected using rabbit anti-Vimentin antibody and HA-BECN1 was detected using rabbit anti-HA antibody. (f) H1299 cells were transfected with pCMV-HA-BECN1 plasmid. Forty-eight hours later, endogenous Vimentin was immunoprecipitated using mouse anti-Vimentin antibody. Western blot was performed to examine the expression of indicated proteins. The K48-linked ubiquitination was detected using K48-linkage specific polyubiquitin antibody. Vimentin was detected using rabbit anti-Vimentin antibody. (g) H1299 cells were transfected with BECN1 siRNAs. Forty-eight hours later, endogenous Vimentin was immunoprecipitated using mouse anti-Vimentin antibody. Western blot was performed to examine the expression of indicated proteins. The K48-linked ubiquitination was detected using K48-linkage specific polyubiquitin antibody. Vimentin was detected using rabbit anti-Vimentin antibody.