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. 2022 Dec 12;42(2):e110553. doi: 10.15252/embj.2021110553

Figure EV2. related to Fig 3. Treatment with the UGCG inhibitor eliglustat promotes TGF‐β signaling and TGF‐β‐induced EMT.

Figure EV2

  1. Levels of p‐SMAD2 and t‐SMAD2 in A549‐VIM‐RFP cells pretreated with eliglustat (1 or 2 μM) for 4 days and then treated with TGF‐β or vehicle control for another 2 days, as shown by immunoblot analysis. GAPDH: loading control.
  2. Effect of eliglustat (2 μM) (pre)treatment for 4 days on SMAD3/SMAD4‐dependent (CAGA)12‐mediated transcriptional GFP reporter expression levels in A549‐VIM‐RFP cells transduced with the CAGA‐GFP lentiviral vector and treated with TGF‐β and/or SB505124 (SB, 1 μM) for the indicated times. The time course of the GFP signal was monitored with an IncuCyte system. The GFP object intensity was normalized to the green intensity at 0 h.
  3. qRT–PCR analysis of TGF‐β target genes, including SMAD7, SERPINE1 and CCN2, in A549‐VIM‐RFP cells pretreated with eliglustat (2 μM) for 6 days and then treated with vehicle control or TGF‐β for 6 h.
  4. CDH1, CDH2, and VIM mRNA levels in A549‐VIM‐RFP cells pretreated with eliglustat (2 μM) for 6 days and then treated with vehicle control or TGF‐β for 6 h.
  5. Alexa Fluor 488 phalloidin staining of F‐actin (green) in A549‐VIM‐RFP cells pretreated with eliglustat (2 μM) for 4 days and then treated with vehicle control or TGF‐β for 48 h. Nuclei were counterstained with DAPI (blue). Images were acquired with confocal microscopy. Scale bar = 50 μm.
  6. Detection of GM1 ganglioside expression in NMuMG cells upon treatment with eliglustat (1 or 2 μM) for 4 days, as analyzed by flow cytometry using Alexa Fluor 488‐conjugated CTB.
  7. Immunoblot analysis of p‐SMAD2 and t‐SMAD2 in NMuMG cells treated with eliglustat (5 or 10 μM) for 4 days and with vehicle control and/or TGF‐β for 1 h. GAPDH: loading control.
  8. Western blot analysis of the expression of the epithelial marker E‐cadherin and mesenchymal marker N‐cadherin in NMuMG cells (pre) treated with eliglustat (5 or 10 μM) for 4 days and with vehicle control and/or TGF‐β for 2 days. Tubulin: loading control.

Data information: TGF‐β was applied at a final concentration of 2.5 ng/ml. All data are expressed as the means ± SD values from three biological replicates (n = 3). *P ≤ 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. In (B), statistical analysis was based on two‐way ANOVA. In (C, D), statistical analysis was based on unpaired Student's t‐test.