epithelial cell medium-plus  (Innoprot Inc)

Journal: Investigative Ophthalmology & Visual Science

Article Title: Insights Into the FOXE3 Transcriptional Network and Disease Mechanisms From the Investigation of a Regulatory Variant Driving Complex Microphthalmia

doi: 10.1167/iovs.66.11.47

Figure Lengend Snippet: Comparative histologic analysis of ocular development in Foxe3 mutants and control samples. ( A ) Representative live view, OCT images, and histologic sections and from adult wild-type mice ( a–c ) and mutant mice ( n ≈ 25 per genotype) illustrate anomalies of increasing severity from a focal anterior subcapsular cataract lesion in Foxe3rv/rv eyes (*) ( d–f ) to cataract (**) ( i ) with extensive irido-lenticular and irido-corneal adhesions and focal fibrotic thickening of the anterior lens capsule (*) ( h , i ) in Foxe3rv/fs ( g–i ) to complex microphthalmia, with corneal clouding (*) ( j , k ), central pit ( arrow ) and athalamia (**) ( j , k ), extensive uveo-corneal adhesions (**) ( l ), and a small, vacuolated, triangular-shaped cataractic lens (*) ( l ) in Foxe3fs/fs . ( B ) Hematoxylin and eosin–stained eye sections of Foxe3 +/+, Foxe3rv/fs , and Foxe3fs/fs mouse embryos from E12.5 to birth (P0) illustrate ocular development ( a–t ). ( a–e ) In Foxe3 +/+ animals, early lens development is seen at E12.5, with the lens vesicle detaching from the surface ectoderm, a defined optic cup, and a neuroepithelial layer forming in the retina ( a ). By E13.5, the lens vesicle is rounder, with early fiber cell differentiation and thickening retina, marking early stratification ( b ). At E14.5, primary lens fibers elongate, the retinal ganglion cell layer becomes visible, and the optic nerve head connection develops ( c ). By E18.5, the lens and retina have mature features, including distinct retinal layers and defined anterior segments like the cornea and ciliary body ( d ). At P0, the lens is fully mature, with organized fiber cells and a defined capsule, while anterior structures like the cornea and iris continue developing ( e ). ( f–j ) In Foxe3rv/fs animals, ocular development appears largely normal, with minor lens fiber vacuolization (*) ( i , j ), which may contribute to the adult cataract phenotype observed in nonnull mice . In some individuals, a small delay in lens detachment can be observed (*) ( g ). ( k–t ) In Foxe3fs/fs animals, initial development appears normal, but by E13.5, the anterior epithelial layer of the lens is disorganized (**) ( l , q ) and a delay in lens detachment is observed, manifesting by a persistent lenticulo-corneal connection (*) ( l , q ). Mild vacuolization and swelling of lens fibers become apparent by E14.5, worsening over time (* in m , r , s , and t ). The lens remains unusually close to the presumptive cornea, giving the appearance of an open lens at the anterior pole (** in s and t ), with possible protein release into the corneal mesenchyme. The anterior epithelial layer gradually disappears, resulting in a microphakic lens ( n , o , s , and t ). Additionally, inconsistent fibrosis is observed within the primary vitreous vascularization, extending from the posterior lens pole to the retina (**) ( n , o , and t ).

Article Snippet: Human lens epithelial cells (HLEpiCs; Innoprot, Derio, Bizkaia, Spain) were cultured in 6-well plates using Epithelial Cell Medium (EpiCM; Innoprot) supplemented with 10% fetal bovine serum (FBS) at 37°C in 5% CO 2 .

Techniques: Control, Mutagenesis, Staining, Cell Differentiation

Journal: Investigative Ophthalmology & Visual Science

Article Title: Insights Into the FOXE3 Transcriptional Network and Disease Mechanisms From the Investigation of a Regulatory Variant Driving Complex Microphthalmia

doi: 10.1167/iovs.66.11.47

Figure Lengend Snippet: Identification and functional analysis of transcription factors binding to Foxe3 regulatory element in lens cells. ( A ) Combined DNA-PD and mass spectrometry analysis in murine CCE cells, CCE-Rx cells, and immortalized HLEpiC. Factors with a significant preference for binding the wild-type oligonucleotide are marked in red ( P < 0.05), while those preferring the mutant oligonucleotide are marked in blue ( P < 0.05). ( B ) RNAscope in situ hybridization shows spatiotemporal expression patterns of candidate genes. At E12.5, Foxe3 expression localizes within the anterior layer of the presumptive lens, while Cnbp , Gabpa , and Usf2 display ubiquitous expression. ( C ) Relative Foxe3 expression in lens epithelial cells from Foxe3+/+ and rv/rv genotypes after inhibition of Cnbp , Usf2 , and Gabpa , as measured by quantitative RT-PCR (* P < 0.05).

Article Snippet: Human lens epithelial cells (HLEpiCs; Innoprot, Derio, Bizkaia, Spain) were cultured in 6-well plates using Epithelial Cell Medium (EpiCM; Innoprot) supplemented with 10% fetal bovine serum (FBS) at 37°C in 5% CO 2 .

Techniques: Functional Assay, Binding Assay, Mass Spectrometry, Mutagenesis, RNAscope, In Situ Hybridization, Expressing, Inhibition, Quantitative RT-PCR