Fig 2: GABARAPL1 knockout leads to an increase in SMAD protein levels by inhibiting their autophagic degradation. (A) Western blotting analysis of SMAD2, SMAD3, and SMAD4 levels in untreated A549c and KO GABARAPL1 cells. (B) mRNA levels of SMAD2, SMAD3, and SMAD4 were measured by qRT-PCR in untreated A549c and KO GABARAPL1 cells. The values calculated by the ??CT method are relative to H3-3A levels, and are expressed as fold change. (C) Western blotting analysis of SMAD2 and SMAD3 in A549c and KO GABARAPL1, and of SMAD4 in ACHNc and KO GABARAPL1 cells, untreated or treated with 50 mM NH4Cl for 2 h. ß-actin was used as a loading control. Protein levels were quantified using Image Lab. (D) Colocalization of LC3 and SMAD2 in A549c cells transfected with pGFP-LC3 vector and immunostained for SMAD2. Colocalization of the autophagosome markers GFP-LC3 and SMAD2 was analyzed using a confocal microscope and the object base method using the “JACoP” plugin (ImageJ software). Scale bar: 5 µm. (E) GST pull-down analysis of the interaction between GABARAPL1 and SMAD2/3 proteins in A549c whole-cell lysates. Whole A549c cell lysate was submitted in a GST pull-down assay with either GST-GABARAPL1 or GST- as a negative control. Data are representative of three independent experiments. (F) P-LISA analysis of GABARAPL1–SMAD2/3 interaction (red) in A549c cells untreated or treated with TGF-ß 5 ng/mL and TNF-a 20 ng/mL for 24 h in the presence or absence of 50 mM NH4Cl for 2 h. A representative image of three independent experiments is shown. The number of red dots per cell was quantified in at least 30 randomly selected cells in each experiment. Scale bar: 5 µm.

Fig 3: GABARAPL1 knockout leads to an induction of EMT. (A) Measurement of the area of A549c and A549 KO GABARAPL1 cells treated, or not, with TGF-ß (5 ng/mL) and TNF-a (20 ng/mL) for 24 h. The cells were stained using rhodamine phalloidin, and the cell areas were measured using a confocal microscope and Image J software. Scale bar: 5 µm. Twenty cells were analyzed per condition, and in three independent experiments. (B) Western blotting analysis of N-cadherin and E-cadherin levels in A549c and KO GABARAPL1 cells untreated or treated with TGF-ß (5 ng/mL) and TNF-a (20 ng/mL) for 24 h. ß-actin was used as loading control. Protein levels were quantified using Image Lab. (C) mRNA levels of CDH1 and CDH2 were measured by qRT-PCR in A549c and A549 KO GABARAPL1 cells, and in ACHNc and ACHN KO GABARAPL1 cells, untreated or treated with TGF-ß 5 ng/mL and TNF-a 20 ng/mL for 24 h. The values calculated by the ??CT method are relative to H3-3A levels, and are expressed as fold change.

Fig 4: GABARAPL1 knockout increases cell proliferation and invasion. (A) Growth rate of A549c and KO GABARAPL1 cells using MTT assay. The data shown are representative of three independent experiments performed in eight replicates. (B) Invasion of A549c, KO GL1 c1 and KO GL1 c3 in modified Boyden chambers. Cell density was estimated via photographs taken using an EVOS XL Core microscope. (C) Migration rates of A549c, KO GL1 c1, and KO GL1 c3 using a scratch wound-healing assay. Cell migration was quantified by the confluence wound study using IncuCyte from 0 to 24 h. (D) Migration rates of A549c, KO GL1 c1, and KO GL1 c3 using modified Boyden chambers. A representative image of each membrane is shown. Six FOVs were randomly selected, and the number of cells that migrated through the membrane was determined.

Fig 5: GABARAPL1 correlates with EMT markers in lung adenocarcinomas. (A) Correlations between ATG8 and EMT-related gene expression in primary lung adenocarcinoma. Correlation circles were built with the two first components (Dim1 and Dim2) of the principal component analysis (PCA). Dim1 and Dim2 explain 32.18% and 21.62 of variability, respectively. PCA was performed with the RNA-Seq data downloaded from the TCGA data portal (372 samples). (B) Boxplots of GABARAPL1 and GABARAP RNA levels according to cadherin 1 (C) and vimentin (V) in the lung carcinoma TCGA database. C and V RNA values were discretized in negative (NEG) and positive (POS), allowing determination of four groups of tumors with increased EMT, from V_NEG/C_POS to V_POS/C_NEG tumors. Indicated p-values were obtained after analysis of variance of GABARAPL1 and GABARAP RNA levels in the four groups. (C) Immunohistochemical analysis of GABARAP and GABARAPL1 expression in lung adenocarcinoma tumors (60 tumors), divided according to the high vimentin immunoreactivity displayed (EMT+, n = 30), or not (EMT-, n = 30), by tumor cells.