Fig 2: BECN1 induction by MIR516A contributed to autophagic response in human BC cells. (A and B) UMUC3-MIR516Ai, UMUC3-Vector, J82-MIR516Ai, and J82-Vector cells were seeded into 6-well plates, and the cells were then starved in 0.1% FBS DMEM for 12 h. Cell lysates were subjected to western blotting for determination of protein as indicated. (C) Western blot was employed to determine the effects of knockdown of PHLPP2 (transient) on BECN1 expression in UMUC3 cells or UMUC3-MIR516Ai stable cells. ACTB or GAPDH were used as an internal loading control. (D-F) Ectopic expression of GFP-BECN1 was detected by western blotting (D), and the cells were then subjected to the soft agar assay (E). The results are expressed as the mean ±SD. An asterisk (*) indicates a significant decrease (p < 0.05) (F). (G) shRNA BECN1#1, shRNA BECN1#2 and shRNA BECN1#4 were stably transfected into J82-MIR516Ai cells, stable transfectants were identified by determination of BECN1 expression and the conversion of LC3-II by western blotting. (H and I) The LC3 puncta formation of indicated cells was observed and images were captured under a microscope. An asterisk (*) indicates a significant decrease as compared with the nonsense control (p < 0.05). (J) Electron microscopy analysis of the autophagosome in the indicated BC cell transfectants. Black arrows, autophagosomes. (K and L) Representative images of colonies of J82-MIR516Ai-shBECN1#2, J82-MIR516Ai-shBECN1#4 and J82-MIR516Ai-Nonsense cells in soft agar assay were captured using a microscope. The results are expressed as the mean ±SD. An asterisk (*) indicates a significant increase compared with the nonsense control (p < 0.05)

Fig 3: PHLPP2-mediated autophagy was involved in MIR516A regulation of the growth of BC cells. (A) UMUC3-MIR516Ai, UMUC3-Vector, J82-MIR516Ai, and J82-Vector cells were serum-starved for 12 h, and the protein levels of LC3-I and LC3-II were assessed by western blotting. (B, C, and H) The LC3 puncta formation of indicated cells was detected by immunofluorescence, and images were acquired using a microscope. (D and I) The LC3-puncta-positive cell rate was calculated as described in the section of “Materials and Methods.” An asterisk (*) indicates a significant change compared with the vector (or nonsense) control group (p < 0.05). (E, F and J) Electron microscopy analysis of autophagosome in the indicated BC cell transfectants. Black arrows, autophagosomes. (G) The protein levels of LC3-I and LC3-II of indicated cells were assessed by western blotting. (K) UMUC3-MIR516Ai, UMUC3-Vector, J82-MIR516Ai, and J82-Vector cells were seeded into each well of 6-well plates and pretreated with BAF (1.0 nM) for 12 h. The cell extracts were subjected to western blotting. (L and M) UMUC3-MIR516Ai, UMUC3-Vector, J82-MIR516Ai, and J82-Vector cells were subjected to soft agar assay with or without the presence of BAF (1.0 nM). Colonies were counted under a microscope and representative images of colonies were also captured (L). The results showed the colonies per 104 cells and are expressed as the mean ±SD (M). An asterisk (*) indicates a significant decrease in comparison to vector transfectants (p < 0.05). An asterisk (**) indicates a significant increase compared with the vehicle control (p < 0.05)

Fig 4: Knockdown of PHLPP2 promoted BECN1 protein degradation in UMUC3-MIR516Ai -Nonsense cells. (A) UMUC3-MIR516Ai-shPHLPP2#2, UMUC3-MIR516Ai -shPHLPP2#5, and UMUC3-MIR516Ai-Nonsense cells were cultured in 6-well plates until the cell density reached 70–80%. Following synchronization in 0.1% FBS DMEM for 12 h, the medium was replaced with 10% FBS DMEM for another 12 h. Then, total RNA was extracted using the TRIzol reagent, and the mRNA expression of BECN1 was detected by qPCR. (B and C) UMUC3-MIR516Ai-shPHLPP2#2, UMUC3-MIR516Ai-shPHLPP2#5, and UMUC3-MIR516Ai-Nonsense cells were subjected to determination of BECN1 protein degradation in the presence of the eukaryote protein synthesis inhibitor, CHX (50 µg/mL) by western blotting (B). The results are expressed as the mean ±SD from three independent experiments. An asterisk (*) indicates a significant decrease in comparison to UMUC3-MIR516Ai-Nonsense cells (p < 0.05) (C). (D) UMUC3-MIR516Ai-shPHLPP2#2 and UMUC3-MIR516Ai-Nonsense cells were pretreated with proteasome inhibitor MG-132 (10 µM) for 12 h, and then subjected to determination of BECN1 protein degradation in the presence of CHX (50 µg/mL) by western blotting. (E) UMUC3 cells expressing GFP-BECN1 or vector control GFP were co-immunoprecipitated with anti-GFP antibody and then subjected to MS analysis. Numbers of peptides identified by MS for BECN1 degradation-related proteins were listed as indicated. (F) Indicated UMUC3 cell transfectants were subjected to western blot for the determination of BECN1 degradation-related proteins. (G) UMUC3 cell transfectant lysates were subjected to Co-IP with an anti-GFP antibody. The binding proteins were determined by western blot. (H) UMUC3-MIR516Ai–Vector and UMUC3-MIR516Ai-Myc-CUL4A cells were subjected to the determination of BECN1 protein degradation in the presence of CHX (50 µg/ml) by western blot. (I) The knockdown of CUL4A in UMUC3-MIR516Ai-shPHLPP2#2 cells and BECN1 expression was assessed by western blot. (J) The BECN1 protein degradation was evaluated in UMUC3-MIR516Ai -shPHLPP2#2-Nonsense, UMUC3-MIR516Ai-shPHLPP2#2-shCUL4A#1 and UMUC3-MIR516Ai -shPHLPP2#2-shCUL4A#2 cells