Fig 2: miR-106a-5p promotes the proliferation and migration of lung adenocarcinoma cells by inhibiting LKB1. (A) Expression levels of LKB1 protein in Calu-3 cells in each group. (B) Expression levels of miR-106a-5p in Calu-3 cells in each group. (C) LKB1 protein expression levels in Calu-3 cells in each group. (D) Cell viability in each group. (E) Comparison of the cell proliferative ability of each group; magnification, x10. (F) Comparison of the cell migratory ability of each group; magnification, x200. (G) Expression levels of autophagy-related proteins in each group. *P<0.05 vs. control or mimic-NC + Vector-NC group; #P<0.05 vs. mimic + Vector-NC group. miR, microRNA; NC, negative control; BECN1, beclin 1; LKB1, liver kinase B1.

Fig 3: miR-106a-5p promotes the proliferation and migration of lung adenocarcinoma cells, and inhibits autophagy. (A) Expression levels of miR-106a-5p in Calu-3 cells in mimic, inhibitor and respective NC groups. (B) Cell viability in each group. (C) Comparison of the cell proliferative ability of each group; magnification, x10. (D) Comparison of the cell migration ability of each group; magnification, x200. (E) Western blot analysis of autophagy-related proteins in each group. *P<0.05 vs. mimic-NC group; #P<0.05 vs. inhibitor-NC group. miR, microRNA; NC, negative control; BECN1, beclin 1.

Fig 4: CaOx crystals induced autophagy in HK-2 cells(A) The formation of GFP-LC3 puncta was analyzed using confocal microscopy after exposure to different concentrations CaOx crystals for 24 h (red arrows). GFP-LC3 dots/cell were quantified; scale bar: 20 µm. (B) A representative immunoblot and quantification analysis of LC3-II and BECN1 as assayed after exposure to different concentrations of CaOx crystals for 24 h. GAPDH was used as a loading control. (C) A representative immunoblot and quantification analysis of LC3-II and BECN1 as assayed after exposure to 4 mM CaOx crystals at various time points. (D) Representative transmission electronic micrographs showed a markedly increased number of autophagic vacuoles after treatment with vehicle or CaOx crystals (4 mM) for 24 h. The number of autophagic vacuoles per 100 µm2 was determined in transmission electron micrographs. White and red arrows indicated autophagosomes and autolysosomes, respectively; scale bar: 2 µm. Data are presented as the mean ± SD from three experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 versus the control group.

Fig 5: Autophagosome dynamic and aggregation(A) Dynamic of autophagosomes in HepG2 and Hep3B cells stably transfected with MAP1LC3B-GFP-RFP-tag. Treatment with 100 nM panobinostat (6 to 72 h) caused a time dependent shift of fluorescence. RFP lightening appeared stronger than green fluorescence at late treatment time. Control cells are shown at time zero of treatment. Panobinostat caused massive cell death after 72 h. (B) Immunoprecipitation of beclin1 in HepG2 and Hep3B after a short time of treatment. Autophagosomes components Atg12, Map1LC3B and UVRAG increased after treatment with 100 nM panobinostat. Ectopic Map1LC3B (GFP) was detected. (C) Quantification of early and late autophagosome vesicles detected by T.E.M. Mean relative amount of vesicles in HepG2 and Hep3B cells ± SD is shown.