Figure 3. Confirmation of effects of PITX2 knockdown on ALDH1A1 and CXCL6 at the protein level. Results shown are with PITX2 siRNA-1 and TM1. A: Representative western immunoblots of ALDH1A1 and CXCL6 in protein extracts of TM cultures exposed to scrambled siRNA and PITX2 siRNA. B-G: Immunofluorescent analysis of ALDH1A1expression in TM cultured cells exposed to scrambled siRNA (B, C) and PITX2 siRNA (D, E). In cells treated with scrambled siRNA (B), ALDH1A1 expression is apparent in cytoplasm and nucleus of most cells. However, decreased expression is evident in many PITX2 siRNA treated cells (D). No immunofluorescence is observed in the negative control (F). DAPI stained cells are also shown (C, E, G). H-S: Immunohistochemical demonstration of ALDH1A1 and CXCL6 expression in the human eye. Cryosections of anterior section of donated globe observed under light microscope (H, K), by immunofluoresence after staining for ALDH1A1 and CXCL6 (I, L, N, O), and after staining with DAPI (J, M, Q, R) are shown. H, I, J: sections stained with anti-ALDH1A1. K, L, M: sections stained with anti-CXCL6. Expression of both ALDH1A1 and CXCL6 in the trabecular meshwork (TM) and stroma (S), and higher expression in the Descemet membrane (DM) are evident. N, Q: highest expression of ALDH1A1 was observed in the epithelium of the cornea (EP). O, R: highest expression of CXCL6 was observed in the iris (I), particularly in the iris epithelium. P, S: negative control shows no immunofluorescent staining. Optical lens magnification 5× (all except O and R), 20× (O and R).