Figure 2.

Knockdown tetraspanin 1 (TSPAN1) promoted molecular characterization of epithelial‐to‐mesenchymal transition state in alveolar epithelial cells. A, Immunofluorescence analysis of subcellular localization for TSPAN1 in A549 cells (green: TSPAN1; red: DiI‐cell membrane red fluorescent probe; blue: 4′,6‐diamidino‐2‐phenylindole [DAPI]), and TSPAN1 was co‐located with DiI in A549 cell. Magnification: 1260X. B, Interference efficiency of siRNA for TSPAN1 was identified in 293T cells by Western blotting. Four siRNA sequences, named si‐TSPAN1‐1, si‐TSPAN1‐2, si‐TSPAN1‐3 and si‐TSPAN1‐4, were designed to aim four target sites for TSPAN1. C, Quantification of the results in panel (B). The data are expressed as the means ± SEM (n = 3), with results representative of three independent experiments. (**P < 0.01 vs negative control for siRNA [NC‐siRNA]). (D) Immunofluorescence staining for TSPAN1 and alpha‐smooth muscle actin (α‐SMA) after transfected with siRNA for TSPAN1 or NC in A549 cells with or without TGF‐β₁treatment (5 ng/mL TGF‐β₁or control) (Green: TSPAN1; Red: α‐SMA; Blue: DAPI). Magnification: 1200X. E, mRNA expression of mesenchymal genes in A549 with or without TGF‐β₁ treatment as defined by qRT‐PCR. The data are expressed as the means ± SEM (n = 3), with results representative of three independent experiments. (*P < 0.05, **P < 0.01, ***P < 0.001 vs control; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs control [NC‐siRNA]; ΔP < 0.05, ΔΔP < 0.01, ΔΔΔP < 0.001 vs control [siRNA‐TSPAN1‐1]). (F,G) Fluorescence activated cell sorting profile showed the CD51/CD61 and CD106 subpopulation in A549 cells with or without TGF‐β₁ treatment. H, Quantification of the results in panel (F) and (G). The data are expressed as the means ± SEM (n = 3), with results representative of three independent experiments. (*P < 0.05, **P < 0.01, ***P < 0.001 vs control; ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 vs control [NC‐siRNA]; ΔP < 0.05, ΔΔP < 0.01, ΔΔΔP < 0.001 vs control [siRNA‐TSPAN1‐1])