Fig 2: 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 3: The GABARAPL1 knockout does not modify autophagic flux. (A) Western blot analysis of GABARAPL1 levels in A549c, ACHNc, and KO GABARAPL1 cells untreated or treated with 50 mM NH4Cl for 2 h. (B) Western blotting analysis of LC3B levels in A549c, ACHNc, and KO GABARAPL1 cells cultured in complete medium and treated, or not, with 50 mM NH4Cl for 2 h. ß-actin was used as a loading control. Protein levels were quantified using Image Lab. (C) GFP-RFP-LC3 puncta analysis in A549c and A549 KO GL1 cells transfected with the ptf-LC3 vector and cultured in complete medium, by confocal microscopy. Each picture is representative of a typical staining observed in 20 fields chosen at random. Red and yellow puncta were counted using ImageJ software (green and red puncta colocalization tool). In each group, 20 cells were randomly selected. Scale bar: 5 m.

Fig 4: 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.

Fig 5: GABARAPL1 knockout leads to an increase in SNAI1 levels. (A) Western blotting analysis of SNAI1 levels in untreated A549c and KO GABARAPL1 cells. (B) Western blotting analysis of SNAI1 levels in A549c 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. Data are represented as the mean ± S.E.M. of at least five independent experiments; p * < 0.05 compared to the control; p-values were calculated using Student’s t-test. (C) mRNA levels of SNAI1 were measured by qRT-PCR in untreated A549c, ACHNc, and KO GABARAPL1 cells. The values calculated by the ??CT method are relative to H3-3A levels, and are expressed as fold change. Data are represented as the mean ± S.E.M. of three independent experiments; p * < 0.05 compared to the control; p-values were calculated using Student’s t-test. (D) mRNA levels of SNAI1 in A549c cells transfected with pCDNA_Control (pCtrl) or A549 KO GL1 c1 cells transfected either with pCtrl or pCDNA-Flag-GABARAPL1-6His (pGL1). The values calculated by the ??CT method are relative to the levels of H3-3A, and are expressed as fold change.