Fig. 2: LEMD3 maintains the contractile phenotype of VSMCs.
a Representative images of immunocytochemical staining for LEMD3 in primary rat VSMCs. Scale bar = 100 µm. n = 3 independent experiments. b Representative images of immunohistochemical staining for LEMD3 in human internal mammary arteries. The region between the dashed lines corresponds to the medial area. Scale bar = 20 µm. n = 3 independent samples. c Representative images of immunofluorescence staining of rat VSMCs transfected with the pEGFP-N1-LEMD3 plasmid or control plasmid. The nuclei were stained with DAPI (blue). Scale bar = 5 µm. This experiment was repeated three times with similar results. d Representative Western blotting and quantification of LEMD3, ACTA2, CNN1, TAGLN in the A7r5 rat VSMC cell line under serum starvation (48 h). GAPDH was used as an internal control. The data are presented as the relative fold changes to control (Ctrl) group (n = 6 independent experiments). e Representative Western blotting and quantification of LEMD3, ACTA2, CNN1, TAGLN in the A7r5 rat VSMC cell line under rapamycin treatment (100 nM, 48 h). GAPDH was used as an internal control. The data are presented as the relative fold changes to vehicle group (n = 6 independent experiments). f Representative Western blotting and quantification of LEMD3, ACTA2, CNN1, TAGLN in the A7r5 rat VSMC cell line under PDGF-BB treatment (10 ng/ml, 48 h). GAPDH was used as an internal control. The data are presented as the relative fold changes to vehicle group (n = 6 independent experiments). g Representative western blotting and quantification of LEMD3 in the lysates of sham-operated and wire-injured mouse carotid arteries at 3 days after surgery. GAPDH was used as an internal control. Data were presented as relative fold change to Sham (n = 4 independent mice). h Representative Western blotting and quantification of LEMD3, ACTA2, CNN1, and TAGLN in the A7r5 rat VSMC cell line transfected with the scrambled siRNA (20 nM) or siRNA targeting Lemd3 (20 nM) for 12 h, followed by serum starvation treatment (48 h). GAPDH was used as an internal control. The data are presented as the relative fold changes to siRNAscramble (n = 4 independent experiments). i Representative Western blotting and quantification of LEMD3, ACTA2, CNN1, and TAGLN in the A7r5 rat VSMC cell line transfected with the scrambled siRNA (20 nM) or siRNA targeting Lemd3 (20 nM) for 12 h, followed by rapamycin treatment (100 nM, 48 h). GAPDH was used as an internal control. The data are presented as the relative fold changes to siRNAscramble (n = 4 independent experiments). j Representative images of phalloidin staining and quantification of F-actin (red) in primary rat VSMCs transfected with the scrambled siRNA (20 nM) or siRNA targeting Lemd3 (20 nM) for 48 h. DAPI staining (blue) indicates the nucleus. Scale bar = 25 µm. n = 3 independent experiments. k Collagen gel contraction assays using primary rat VSMCs transfected with the scrambled siRNA (20 nM) or siRNA targeting Lemd3 (20 nM) for 48 h. n = 3 independent experiments. l EdU incorporation assays of primary rat VSMCs transfected with the scrambled siRNA (20 nM) or siRNA targeting Lemd3 (20 nM) for 48 h. n = 6 independent experiments. Error bars indicate s.e.m. Statistical analysis was performed using two-sided unpaired Student’s t test for (d-g), two-way ANOVA followed by Bonferroni’s multiple comparisons test for (h) and (i), the χ2 test for (j) and the Mann‒Whitney U test for (k) and (l).
