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Impact of Nrf2 on ROS Levels and E-Cadherin Expression in A549 Cells Under TGF-β Treatment

This study investigates the role of Nrf2 in regulating reactive oxygen species (ROS) levels and E-cadherin expression in A549 cells subjected to TGF-β treatment. Utilizing shRNA methods, we observed changes in ROS and E-cadherin levels that correlate with Nrf2 expression. Supplemental figures illustrate key data, including cellular responses under different experimental conditions. Our findings suggest that modulation of Nrf2 activity influences the cellular redox state and epithelial-mesenchymal transition markers, providing insights into potential therapeutic targets in cancer biology.

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Impact of Nrf2 on ROS Levels and E-Cadherin Expression in A549 Cells Under TGF-β Treatment

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  1. Vimentin FLAG/Luc/#1 FLAG/Keap1/#1 FLAG/Luc/#1 FLAG/Keap1/#1 B Supplemental Figure 1 A

  2. Supplemental Figure 2 GSH 25,000,000 20,000,000 Relative Intensity 15,000,000 10,000,000 5,000,000 0 Control Nrf2 shRNA/#2 Levels of ROS A B Control Nrf2 shRNA Cell Number C400 Control shRNA Nrf2 shRNA/#2 C Number x 10

  3. Nrf2 shRNA #2 Control shRNA pAKT AKT C-Jun Cyclin D1 GAPDH Supplemental Figure 3

  4. A549 cells Control lentivirus shRNA Lentivirus Nrf2 shRNA clone 3 Lentivirus Nrf2 shRNA clone 6 Nrf2 Cell Number GAPDH Control Clone 3 Days Lentivirus Nrf2 shRNA clones Supplemental Figure 4 B Clone 6

  5. Supplemental Figure 5 Nrf2 shRNA Control shRNA

  6. 0 hr 24 hr 24 hr 5 ng/ml TGF- SB431532 + TGF- Supplemental Figure 6 B A Control retrovirus shRNA Nrf2 siRNA in pSilencer Retrovirus Nrf2 shRNA #2 Control siRNA in pSilencer O hr 24hr 24 hrs C

  7. 24 hrs + TGF- 24 hrs TGF- + SIS3 Supplemental Figure 7

  8. Supplemental Figure 8 Micro-Taur schematic diagram “A” side media ports Cell loading port “B” side media ports • 6 micron posts • 8 micron posts • 10 micron posts • 12 micron posts Post-containing chambers Insert illustrating Post Region (12 micron)

  9. Supplemental Figure 9 2500 2000 1500 TGF-1 pg/ml 1000 500 0 Control shRNA Nrf2-shRNA #2

  10. A B Loss of Nrf2 expression TGF β TGF β E-Cadherin ROS ROS Phosphatases P P P P P P Phosphatases PP PP PP PP MH2 MH2 MH2 MH2 MH1 MH1 MH1 MH1 MH2 MH1 R-Smad R-Smad R-Smad R-Smad R-Smad pR-Smad pR-Smad pR-Smad Smad4 Smad4 Smad4 Nrf2 Smad target genes Smad target genes Nrf2 Nrf2 Slug Slug Smad target genes Supplemental Figure 10 E-Cadherin

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