Journal: Investigative Ophthalmology & Visual Science
Article Title: A Cross-Species Hydroquinone Model Replicates Smoking-Related Corneal Endothelial Oxidative Injury and Enables Therapeutic Screening of FGF10
doi: 10.1167/iovs.67.3.12
Figure Lengend Snippet: HQ exposure triggers concentration-dependent transcriptional responses in hCEnCs. ( A ) Heatmap of all differentially expressed genes, averaged across triplicates, demonstrates progressive up- and downregulation with increasing HQ concentrations. ( B ) GSEA using REACTOME_2022 comparing 150 µM and 0 µM HQ reveals activation of integrated stress response and antioxidant pathways. ( C ) Nrf2 pathway heatmap. Blue boxes mark glutathione biosynthesis genes ( Gclc , Gclm ). Purple boxes mark phase II detoxification and redox genes ( Nqo1 , Srxn1 , Txnrd1 , Gstp1 , Pgd ). Orange boxes mark redox buffering/transport genes ( Slc7a11 , Sod1 , Sod2 , Hmox1 ). ( D , E ) Western blot analysis of ( D ) Hmox1 and ( E ) Sod1, selected as representative Nrf2-regulated antioxidant proteins, shows statistically significant increases in expression in hCEnCs treated with HQ at 100 µM and 150 µM. ( F ) Hippo–Yap pathway heatmap. Gray boxes mark core transcriptional effectors ( Yap1 , Tead1 , Tead2 , Tead3 , Tead4 ). Pink boxes mark upstream kinases and scaffolds ( Lats2 , Mob1a , Sav1 , Stk4 ). Green boxes mark membrane-associated and regulatory proteins ( Amotl1 , Amotl2 , Nf2 , Wwc1 , Frmd6 ). ( G ) Western blot analysis of pYap and Yap shows increased pYap/Yap ratio levels at 150 µM compared to control. ( H ) Representative immunofluorescence images show DAPI ( blue ), YAP ( green ), and merged channels for control and 150 µM HQ treatment, with a higher-magnification inset of the merged image. Scale bar : 100 µM. Statistical significance: * P < 0.05, ** P < 0.01, *** P < 0.001.
Article Snippet: Proteins were separated on NuPAGE Bis-Tris Mini Protein Gels (4%–12%), transferred to PVDF membranes using the Trans-Blot Turbo Transfer System (Bio-Rad), blocked with 3% Blotting-Grade Blocker, and incubated overnight at 4°C with primary antibodies against GAPDH (1:200; cat. 2118S, Cell Signaling Technologies), β-actin (1:200; cat. 66009-1-Ig, Proteintech, Chicago, Illinois, USA), Sod1 (1:200; cat. 37385T, Cell Signaling Technologies), Hmox1 (1:200; cat. 26416T, Cell Signaling Technologies), N-cadherin (1:200; cat. 13116T, Cell Signaling Technologies), β-catenin (1:200; cat. sc-7199, Santa Cruz Biotechnology), Yap (1:200; cat. sc-15407, SCBT), or phospho-Yap (1:200; cat. 13008S Cell Signaling Technologies).
Techniques: Concentration Assay, Activation Assay, Western Blot, Expressing, Membrane, Control, Immunofluorescence
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