Fig. 2

Knockdown of CSMD1 promoted cell migration and FN1 secretion in fibroblasts. (A) qRT-PCR was performed to compare the CSMD1 mRNA expression in fibroblasts transfected with Lenti-shRNA-CSMD1 (shCSMD1) and Lenti-GFP (shNC). All experiments were performed in triplicate and the data were shown as mean ± SD, *p < 0.05. (B) Immunofluorescence was performed to confirm the knockdown of CSMD1 protein expression in the CSMD1-silenced fibroblasts. Scale bar: 200 μm. (C) Histogram showing fluorescence intensity of CSMD1 in the shNC and shCSMD1 fibroblasts from the IF photos taken and analyzed using the NIS-Elements D software. (D-E) Transwell assays were performed to detect the migration of CSMD1-silenced fibroblasts. Quantification of numbers of migrated cells per field was presented as mean ± SD from three independent experiments in the right panel. Scale bar: 200 μm, **p < 0.01. (F-G) Wound healing assays were performed to detect the migration of CSMD1-silenced fibroblasts. The wound area at 0 h was set as 100%. Quantification of the healing rate was presented as mean ± SD from three independent experiments in the right panel. Scale bar: 200 μm, **p < 0.01. (H-I) qRT-PCR and western blot were performed to measure the ACTA2, COL1 and FN1 mRNA and protein levels respectively in the shNC and shCSMD1 fibroblasts. The results showed significantly upregulated mRNA levels of ACTA2, COL1 and FN1 upon CSMD1 knockdown, while only increased expression of FN1 at the protein level. *p < 0.05