Journal: International Journal of Molecular Sciences

Article Title: 7-Ketocholesterol Promotes Retinal Pigment Epithelium Senescence and Fibrosis of Choroidal Neovascularization via IQGAP1 Phosphorylation-Dependent Signaling

doi: 10.3390/ijms241210276

Figure Lengend Snippet: 7-ketocholesterol (7KC) does not promote epithelial mesenchymal transition (EMT) or death of the retinal pigment epithelium (RPE). Western blots of ( A ) mesenchymal markers, fibroblast activation protein (FAP) and alpha smooth muscle actin (αSMA), ( B ) RPE specific markers, bestrophin 1 (BEST1), RPE65, CRALBP, PDGFRβ and integrin β4, ( C , D ) caspase 1 and densitometry; ( E ) TUNEL staining (the scale of the bars: 100 µm; red: tunnel positive cells), ( F ) hRPE viability measured by MTT assay, ( G ) western blot of BCL2, and ( H ) real time PCR of BCL-xL mRNA in HRPE treated with HPBCD or 7KC for 48 h or in untreated HRPE (* p < 0.05, ** p < 0.01 vs. HPBCD; n = 4–6).

Article Snippet: Experiments performed with ECIS demonstrated that control-treated ARPE-19 or Lonza hRPE had similar resistances (2000–2200 ohms·cm 2 ). hRPE was treated with 7-ketocholesterol (7KC, 10 µM, Avanti Polar Lipids, Birmingham, AL, USA) or volume-matched solvent control, 2-hydroxypropyl-β-cyclodextrin (HPBCD, MilliporeSigma, St. Louis, MO, USA) for 24 or 48 h. In some experiments, the hRPE was pretreated with rapamycin (10 nM, Selleckchem, Pittsburgh, PA, USA), Go6983 (10 μM, Tocris Bioscience, Minneapolis, MN, USA), or an equal volume of vehicle control DMSO for 30 min prior to incubation with 7KC or HPBCD.

Techniques: Western Blot, Activation Assay, TUNEL Assay, Staining, MTT Assay, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: 7-Ketocholesterol Promotes Retinal Pigment Epithelium Senescence and Fibrosis of Choroidal Neovascularization via IQGAP1 Phosphorylation-Dependent Signaling

doi: 10.3390/ijms241210276

Figure Lengend Snippet: 7-ketocholesterol (7KC) induces RPE cell cycle arrest at mitosis, senescence and senescence associated secretory phenotype (SASP). Western blots of ( A ) of phospho-Histone H3 (Ser10), ( A – D ) p16, p21 and LaminB1; ( E , F ) β-galactosidase (β-gal) (the scale of bars: 100 µm); western blots of ( G , H ) phosphorylation of NF-κB (p-NF-κB), total NF-κB, ( I , J ) IL-1β, ( I , K ) IL-6 and ( I , L ) VEGF in HRPE cells treated with HPBCD or 7KC for 48 h (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. HPBCD; ( A , E , G , I ): representative images; ( B – D , F , H , J – L ): quantification results; n = 4–6).

Article Snippet: Experiments performed with ECIS demonstrated that control-treated ARPE-19 or Lonza hRPE had similar resistances (2000–2200 ohms·cm 2 ). hRPE was treated with 7-ketocholesterol (7KC, 10 µM, Avanti Polar Lipids, Birmingham, AL, USA) or volume-matched solvent control, 2-hydroxypropyl-β-cyclodextrin (HPBCD, MilliporeSigma, St. Louis, MO, USA) for 24 or 48 h. In some experiments, the hRPE was pretreated with rapamycin (10 nM, Selleckchem, Pittsburgh, PA, USA), Go6983 (10 μM, Tocris Bioscience, Minneapolis, MN, USA), or an equal volume of vehicle control DMSO for 30 min prior to incubation with 7KC or HPBCD.

Techniques: Western Blot

Journal: International Journal of Molecular Sciences

Article Title: 7-Ketocholesterol Promotes Retinal Pigment Epithelium Senescence and Fibrosis of Choroidal Neovascularization via IQGAP1 Phosphorylation-Dependent Signaling

doi: 10.3390/ijms241210276

Figure Lengend Snippet: Inhibition of mTOR signaling inhibits 7-ketocholesterol mediated RPE senescence and SASP. Western blots of ( A ) phosphorylation of mTOR (p-mTOR), total mTOR, phosphorylation of S6K1 (p-S6K1), total S6K1, ( B ) phospho-Histone H3 (Ser10), ( B , C ) p16, ( B , D ) p21, ( E ) phospho-NF-κB (p-NF-κB) and total NF-κB, ( F , G ) IL-1β, ( F , H ) VEGF and ( F , I ) IL-6 in hRPE pretreated with Control or Rapamycin (10 nM) for 30 min prior to incubation with HPBCD or 7KC for 48 h (** p < 0.01 and *** p < 0.001 vs. HPBCD of Control; ††† p < 0.001 vs. 7KC of Control; n = 4–6).

Article Snippet: Experiments performed with ECIS demonstrated that control-treated ARPE-19 or Lonza hRPE had similar resistances (2000–2200 ohms·cm 2 ). hRPE was treated with 7-ketocholesterol (7KC, 10 µM, Avanti Polar Lipids, Birmingham, AL, USA) or volume-matched solvent control, 2-hydroxypropyl-β-cyclodextrin (HPBCD, MilliporeSigma, St. Louis, MO, USA) for 24 or 48 h. In some experiments, the hRPE was pretreated with rapamycin (10 nM, Selleckchem, Pittsburgh, PA, USA), Go6983 (10 μM, Tocris Bioscience, Minneapolis, MN, USA), or an equal volume of vehicle control DMSO for 30 min prior to incubation with 7KC or HPBCD.

Techniques: Inhibition, Western Blot, Incubation

Journal: International Journal of Molecular Sciences

Article Title: 7-Ketocholesterol Promotes Retinal Pigment Epithelium Senescence and Fibrosis of Choroidal Neovascularization via IQGAP1 Phosphorylation-Dependent Signaling

doi: 10.3390/ijms241210276

Figure Lengend Snippet: Inhibition of mTOR by rapamycin prevents 7-ketocholesterol (7KC)-mediated barrier compromise of RPE. ( A , B ): Co-immunoprecipitation of cadherin and β-catenin, ( C ) immunostaining of junctional protein ZO-1 (the scale of bars: 100 µm; red: ZO-1 stain), and ( D ) transepithelial resistance (TER) of hRPE treated with HPBCD or 7KC or without control or experimental treatment (untreated) for 48 h before being processed for different assays (( A ), representative gel images, ( B ), quantification results; ** p < 0.01, *** p < 0.001 vs. HPBCD).; ( E , F ) Electric Cell-Substrate Impedance Sensing (ECIS) of hRPE treated with Control+HPBCD, Control+7KC, Rapamycin+HPBCD, Rapamycin+7KC (( E ), relative resistance measurements and ( F ), quantification of area under the curve (AUC) of 24 h resistance measurements; ††† p < 0.001 vs. Control+HPBCD, ### p < 0.001 vs. Control+7KC; n = 4–6).

Article Snippet: Experiments performed with ECIS demonstrated that control-treated ARPE-19 or Lonza hRPE had similar resistances (2000–2200 ohms·cm 2 ). hRPE was treated with 7-ketocholesterol (7KC, 10 µM, Avanti Polar Lipids, Birmingham, AL, USA) or volume-matched solvent control, 2-hydroxypropyl-β-cyclodextrin (HPBCD, MilliporeSigma, St. Louis, MO, USA) for 24 or 48 h. In some experiments, the hRPE was pretreated with rapamycin (10 nM, Selleckchem, Pittsburgh, PA, USA), Go6983 (10 μM, Tocris Bioscience, Minneapolis, MN, USA), or an equal volume of vehicle control DMSO for 30 min prior to incubation with 7KC or HPBCD.

Techniques: Inhibition, Immunoprecipitation, Immunostaining, Staining, Electric Cell-substrate Impedance Sensing

Journal: International Journal of Molecular Sciences

Article Title: 7-Ketocholesterol Promotes Retinal Pigment Epithelium Senescence and Fibrosis of Choroidal Neovascularization via IQGAP1 Phosphorylation-Dependent Signaling

doi: 10.3390/ijms241210276

Figure Lengend Snippet: PKC-dependent serine phosphorylation is involved in 7KC-mediated RPE senescence and SASP. Western blots of ARPE-19 pretreated with control or Go6983 prior to incubation with HPBCD or 7KC and measured for ( A ) p-PKC, serine phosphorylation of IQGAP1 and total IQGAP1, ( B ) p-mTOR, total mTOR, p-S6K1 and total S6K1, and ( C ) p21, VEGF, and IL-1b ( n = 4–6).

Article Snippet: Experiments performed with ECIS demonstrated that control-treated ARPE-19 or Lonza hRPE had similar resistances (2000–2200 ohms·cm 2 ). hRPE was treated with 7-ketocholesterol (7KC, 10 µM, Avanti Polar Lipids, Birmingham, AL, USA) or volume-matched solvent control, 2-hydroxypropyl-β-cyclodextrin (HPBCD, MilliporeSigma, St. Louis, MO, USA) for 24 or 48 h. In some experiments, the hRPE was pretreated with rapamycin (10 nM, Selleckchem, Pittsburgh, PA, USA), Go6983 (10 μM, Tocris Bioscience, Minneapolis, MN, USA), or an equal volume of vehicle control DMSO for 30 min prior to incubation with 7KC or HPBCD.

Techniques: Western Blot, Incubation

Journal: International Journal of Molecular Sciences

Article Title: 7-Ketocholesterol Promotes Retinal Pigment Epithelium Senescence and Fibrosis of Choroidal Neovascularization via IQGAP1 Phosphorylation-Dependent Signaling

doi: 10.3390/ijms241210276

Figure Lengend Snippet: 7KC promotes CNV fibrosis through IQGAP1 serine phosphorylation. ( A ) Representative choroidal flat mounts showing laser induced lesions labeled with lectin (Green) and αSMA (red) (the scale of bars: 100 µm) and ( B ) Lesion volumes measured by lectin or αSMA staining 2 weeks after laser and intravitreal 7KC injections in CRISPR/IQ vs. wildtype mice (* p < 0.05).

Article Snippet: Experiments performed with ECIS demonstrated that control-treated ARPE-19 or Lonza hRPE had similar resistances (2000–2200 ohms·cm 2 ). hRPE was treated with 7-ketocholesterol (7KC, 10 µM, Avanti Polar Lipids, Birmingham, AL, USA) or volume-matched solvent control, 2-hydroxypropyl-β-cyclodextrin (HPBCD, MilliporeSigma, St. Louis, MO, USA) for 24 or 48 h. In some experiments, the hRPE was pretreated with rapamycin (10 nM, Selleckchem, Pittsburgh, PA, USA), Go6983 (10 μM, Tocris Bioscience, Minneapolis, MN, USA), or an equal volume of vehicle control DMSO for 30 min prior to incubation with 7KC or HPBCD.

Techniques: Labeling, Staining, CRISPR