HSF4 regulates lens-specific bead filaments Bfsp1 and Bfsp2. (A) Real-time PCR to determine mRNA levels of Bfsp1 and Bfsp2 in lenses of newborn and adult wild-type and Hsf4 knockout mice. (B) PCR analysis of Bfsp2 gene deletion in Hsf4-/-, Hsf4+/-, 129S3 and C57BL/6J mice using primers from the deleted region of the Bfsp2 gene in 129S3 mice. The results suggest that there is at least one copy of the C57BL/6J Bfsp2 allele in the Hsf4-/- mouse. (C) Real-time PCR analysis of mRNA levels of Bfsp1 and Bfsp2 after transfection of SRA01/04 with HSF4b, shRNA for HSF4, or HSF4b plus shRNA for HSF4. (D) Promoter sequence alignment of Bfsp1 and Bfsp2. 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) Bfsp1 and Bfsp2 gene promoters were PCR-amplified from chromatin immunoprecipitation-enriched DNA from human lens epithelial cells (SRA01/04) transfected with HSF4b. (F) Relative luciferase activities of the promoter-luciferase constructs (Bfsp1-luc or Bfsp2-luc) after 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.