Figure 1. Effects of MYOC Mutations on Expression of IL1A and other Genes Associated with Glaucoma in Transient Transfections Cultures of TM-1 cells were transiently transfected with pCMV6 expression constructs containing wild-type (WT) or mutant (Q368X, Y437H) MYOC genes. A set of cells was also transfected with empty vector (EV) control for comparison. After 24 h incubation, cell lysates were collected for western blotting or for extraction of RNA and RT–PCR analysis. A:Lanes of an SDS-polyacrylamide gel were equally loaded with aliquots of each sample (20 ug) and proteins were separated by electrophoresis. Western blotting was performed with anti-MYOC antibody. The electrophoretic mobility of molecular weight size standards is indicated. B: Equal aliquots of total RNA were analyzed by qRT-PCR for IL1A mRNA, and normalized to ACTB, which served as an internal control. Results obtained using MYOC expression constructs were compared to EV controls. C: Equal aliquots of total RNA described in (B) were also analyzed by real-time reverse transcription-polymerase chain reaction (RT–PCR) for IL1B, IL6, TGFB2, ACTA2 and FOXO1 mRNAs, and normalized to ACTB, which served as an internal control. Results obtained using MYOC expression constructs were compared to EV controls. Mutant MYOC overexpression increased mRNA levels for IL1B (Q368X*), ACTA2 (Q368X** and Y437H*) and FOXO1 (Q368X** and Y437H**); wild-type MYOC overexpression decreased mRNA levels for IL1B* and IL6*, TGFB2* and FOXO1**; *p<0.05; **p<0.001; n=3.