Suppl. Figure 3 A H1975 Atg7 shRNA 3 MOI Atg7 shRNA 6 MOI Scramble 3 MOI Scramble 6 MOI 100 H1975 ATG7 78 kDa 75 GAPDH 37 kDa 37 Suppl. Figure 3. Targeted Knock Down of ATG7 gene modulates TGFβ1-induced EMT in A549 and H1975 cells. (A) ATG7 gene was knocked down in both cell lines using lentivirus carrying the specific siRNA against the target gene. Different MOIs (3 and 6) of lentiviral particles were used in order to identify the most efficient one to knock down the ATG7 gene. Scramble siRNA (without the specific sequence for ATG7 gene) was used as control.

Suppl. Figure 3 B Scramble shRNA-ATG7 C0 24 36 24 36 C0 24 36 24 36 ATG7 78 kDa p-Smad2 60 kDa C0 24 36 24 36 Scramble C0 24 36 24 36 shRNA-ATG7 Total-Smad2 60 kDa LC3β-I 18 kDa LC3β-II 16 kDa GAPDH 37 kDa E-Cadherin 135 kDa Vimentin 57 kDa TGF-β1 Media TGF-β1 Media B 100 75 20 15 150 100 50 Suppl. Figure 3. (B) H1975 cells (ATG7 KD) were treated with TGFβ1 for 24 and 36 hrs. The abundance of EMT, Smad signaling and autophagy markers were evaluated using western blotting. Figures show the representative western blotting bands. GAPDH was used as the loading control. 50 50 37

Suppl. Figure 3 C Suppl. Figure 3. (C) The blots for vimentin were quantified for both cells compared to their correspondent controls using densitometry software Alpha Ease FC. The expression level of vimentin is significantly lower in ATG7 KD cells compared to the scramble cells. Graphs were prepared by GraphPad Prism. P-value ≤ 0.05 and lower were considered as statistically significant.

Suppl. Figure 3 D Vimentin-AF647 GFP-LC3 Merge DAPI Scramble-Control Suppl. Figure 3. (D) Immunocytochemistry results showed that the ATG7 KD H1975 cells have a lower signal for vimentin and less level of LC3B-II puncta as compared to their scramble counterparts after treatment with TGFβ1 (5ng/µl) for 48hrs. Yellow arrows show the LC3 punctuate and highly expressed vimentin in scramble cells treated with TGFβ1. Representative immunofluorescent images of LC3B-II and activated lysosomes shown (scale bars 20μm). Scramble-TGFβ1

Suppl. Figure 3 (Continued) Vimentin-AF647 GFP-LC3 Merge DAPI 20 µm Atg7 shRNA-Control Suppl. Figure 3. (D) (Continued) shRNA-TGFβ1