γ-crystallin expression during lens development. (A) Lens weight in 8-week-old wild-type and Hsf4 knockout mice. Ten wild-type lenses and 16 knockout lenses were used to calculate the average weight. (B) mRNA levels of all γ-crystallins were analyzed using quantitative RT-PCR. mRNA isolated from lenses in newborns and 8-week-old mice (C) Real-time PCR analysis of mRNA levels of γS-crystallin using specific primers. Total RNA was isolated from human lens epithelial cells (SRA01/04) transfected with HSF4b, shRNA for HSF4, or HSF4b plus shRNA for HSF4 respectively. (D) Promoter sequence alignment of seven γ-crystallins. Sequences identical to the classic heat-shock element (HSE) sequence are shown in red. Asterisks indicate the key nucleotides essential for heat-shock factor binding. (E) CRYGS gene promoter was PCR-amplified from chromatin immunoprecipitation-enriched DNA from human lens epithelial cells (SRA01/04) transfected with HSF4b. (F) Relative luciferase activity of the promoter-luciferase construct (CRYGS-luc) by transfection with HSF4a or HSF4b in human lens epithelial cells (SRA01/04). The transfections were performed in hexaplets and the Renilla luciferase plasmid was used as normalization control. The results are shown as means with standard deviations. The classic HSE-luc was used as a positive control.