Fig. 2. Tmod3 knockdown by shRNA results in altered cell morphology and decreased fluorescence intensity of F-actin at lateral membranes. (A) Transfection of pEGFP-H1RNAi encoding a hairpin sequence directed against Tmod3 results in decreased Tmod3 protein levels compared to transfection of pEGFP-H1RNAi or pEGFP-H1RNAi encoding mismatched target sequence. (B-E) XZ and (F-I) XY confocal sections stained with rhodamine-phalloidin for F-actin (C,G) shown in red in merges (B,F), and with antibodies against Tmod3 (D,H) shown in green in merged images (B,F), with transfected cells expressing GFP (E,I) shown in blue in merged images (B,F). (J-L) Quantification of cell morphology and total F-actin staining intensity shows that Tmod3 knockdown results in (J) decreased cell height, (K) increased cross-sectional area and, (L) decreased intensity of F-actin fluorescence when compared to untransfected cells in the same field of view, or to cells transfected with mismatched target sequence. (M-O) Line scans demonstrate that Tmod3 knockdown results in decreased F-actin fluorescence intensity at lateral membranes compared with untransfected cells or cells transfected with mismatched target sequence. (M,N) Representative line scans of lateral membrane F-actin fluorescence intensity and peak spacing in shRNA or mismatch transfected or in untransfected cells in the same field of view; 3.5 pixels per µm for both M and N. (O) Quantification of F-actin fluorescence intensity at lateral membranes from the area under peaks in line scans. Results are mean ± s.e.m.; ^*P<0.05; ^**P<0.005.