Fig. 4

NR2F2 regulated the expression of SASP in epithelial cells and influenced fibroblast activation. (A-C) The mRNA expression levels of IL1B and TGFB1 in A549 (A), MLE-12 (B), and BEAS2B (C) cells treated with 0.02 U/ml bleomycin for an additional 72 h after transfection with control or NR2F2 overexpression plasmid for 48 h were quantified using qRT-PCR. (D-F) The mRNA expression levels of IL1B and TGFB1 in A549 (D), MLE-12 (E), and BEAS2B (F) cells stably expressing control or NR2F2 knockdown plasmids were quantified using qRT-PCR. (G) Schematic of the experimental design. The CM from A549 cells after treatment was collected and utilized for the cultivation of MRC-5 cells. (H) After transfection of A549 cells with either control or NR2F2 overexpression plasmids for 48 h, they were treated with 0.02 U/ml of bleomycin for an additional 72 h. Subsequently, the medium was replaced with fresh culture medium to culture for another 48 h, and the supernatant was collected and used to culture MRC-5 cells for 48 h. The protein expression levels of Fibronectin, COL1A1, and α-SMA in MRC-5 cells were measured using WB. (I) The culture medium of A549 cells stably expressing either control or NR2F2 knockdown plasmids was replaced with fresh medium and further incubated for 48 h. After that, the supernatant was collected and utilized to culture MRC-5 cells for 48 h. WB analysis was performed to measure the protein expression levels of Fibronectin, COL1A1, and α-SMA in MRC-5 cells. (J) Schematic diagram of the experimental design for the transwell co-culture system. (K) After transfection of A549 cells with either control or NR2F2 overexpression plasmids for 48 h, they were treated with 0.02 U/ml of bleomycin for an additional 72 h. Subsequently, MRC-5 cells were seeded in the upper transwell chamber and co-cultured with A549 cells for 48 h. The protein expression levels of Fibronectin, COL1A1, and α-SMA in MRC-5 cells were measured using WB. (L) MRC-5 cells were seeded in the upper transwell chamber and co-cultured with A549 cells stably expressing either control or NR2F2 knockdown plasmids in the lower transwell chamber for 48 h. WB analysis was performed to measure the protein expression levels of Fibronectin, COL1A1, and α-SMA in MRC-5 cells. (M, O) The transwell assay and collagen contraction assay were conducted to evaluate the invasive (M) and activation (O) capacities of MRC-5 cells after treatment, respectively, following the same experimental design as depicted in Fig. 4H. (N, P) The transwell assay and collagen contraction assay were performed to evaluate the invasive (N) and activation (P) capacities of MRC-5 cells after treatment, respectively (same experimental design as shown in Fig. 4I). Data are shown as the mean ± SD. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001