Fig. 5

A candidate lncRNA is able to attenuate TGF-β1-induced fibroblast differentiation. Human primary fibroblasts were transfected with esiRNA targeting two candidate lncRNAs (AC093850.2 and RP11-626H12.2), a transcript not expressed in human cells (Evf-2) and a lncRNA from a different module not predicted to influence fibroblast differentiation (RP1-122P22.2). Each experiment consisted of nine technical and three biological replicates. a Cells were dispensed into 384 well plates, reverse transfected with esiRNAs, incubated for 2 days knock-down and then stimulated or not with TGF-β1 for 24 h. Images were acquired using a MetaXpress Micro × 2 objective and cells identified using the nuclei stain Hoechst, and segmented using MetaXpress software. Data processed in Excel and Prism7. b The protocol was identical to that of A, but the cell were stained with αSMA antibody after fixation, and imaged using the × 20 objective. Positive CAFs were identified on the formation of de novo αSMA-positive stress fibres and morphological changes using MetaXpress Custom Module Editor. **p < 0.05, ***p < 0.01, ****p < 0.001. Only comparisons between groups reaching statistical significance are indicated. c Microscope images of unstimulated, control Evf-2 knock-down cells. d TGF-β1-stimulated, control Evf-2 knock-down cells. The white * indicates a transformed CAF with both morphological and αSMA positive fibres. e Unstimulated, AC093850.2 knock-down cells. f TGF-β1-stimulated, AC093850.2 knock-down cells. The white + indicates a cell counted as a CAF with a partial transformation