Fig 2: TGF-β1 stimulates SPARC expression in KGN and primary hGL cells. A KGN cells were treated with 10 ng/mL TGF-β1 for different periods, and the mRNA levels of SPARC were examined by RT-qPCR. The level of SPARC mRNA at each time point was normalized to the GAPDH mRNA level at the same time point. B KGN cells were treated with 10 ng/mL TGF-β1 (T) for 6, 12, and 24 h, and the protein levels of SPARC were examined by western blot. C and D KGN cells were treated with 5 or 10 ng/mL TGF-β1 for 6 h. The SPARC mRNA levels (C) and protein levels (D) were examined by RT-qPCR and western blot, respectively. E Primary hGL cells were treated with 5 ng/mL TGF-β1 (T) for 6, 12, and 24 h, and the protein levels of SPARC were examined by western blot. The results are expressed as the mean ± SEM of at least three independent experiments. Values that are statistically different from one another (p < 0.05) are indicated by different letters

Fig 3: Effects of SPARC on the epithelial-mesenchymal transition of 16HBE cells. (A) SPARC mRNA was measured via RT-qPCR in 16HBE cells treated with LV-SPARC for 24 h. (B) mRNA expression levels of E-cadherin and Vimentin were measured via RT-qPCR. (C) Protein expression levels of E-cadherin and Vimentin were measured by western blotting. Three independent experiments were conducted for each treatment. The data are expressed as the mean ± SD. Data were analyzed using a one-way ANOVA. *P<0.05, **P<0.01, ***P<0.001. LV, lentivirus; SPARC, secreted protein acidic rich in cysteine; RT-qPCR, reverse transcription-quantitative PCR; NeC, negative control group.

Fig 4: SPARC is the target gene for miR-29a-3p. (A) The predicted miR-29a-3p binding sites in the SPARC 3′UTR. (B) The direct regulation of miR-29a-3p to SPARC was determined using dual-luciferase reporter gene assays. (C) miR-29a-3p and (D) SPARC mRNA expression as measured via reverse transcription-quantitative PCR in miR-29a-3p mimic-treated 16HBE cells. Three independent experiments were conducted for each treatment. The data are expressed as the mean ± SD. Data were analyzed using one-way or two-way ANONA as applicable.**P<0.01, ****P<0.0001. UTR, untranslated region; WT, wild-type; MUT, mutant; SPARC, secreted protein acidic rich in cysteine; miR, microRNA; NeC, negative control group.

Fig 5: TGF-β1-induced Slug expression is required for the induction of SPARC. A KGN cells were treated with 5 ng/mL TGF-β1 (T) for 1, 3, and 6 h, and the protein levels of Snail and Slug were examined by western blot. B Primary hGL cells were treated with 5 ng/mL TGF-β1 (T) for 6 h, and the protein levels of Snail and Slug were examined by western blot. C and D KGN cells were transfected with 50 nM control siRNA (si-Ctrl), Snail siRNA (si-Snail) (C) or Slug siRNA (si-Slug) (D) for 48 h, and then treated with 5 ng/mL TGF-β1 (T) for 6 h. The SPARC protein levels were examined by western blot. E KGN cells were transfected with 50 nM control siRNA (si-Ctrl) or SPARC siRNA (si-SPARC) for 48 h. The Snail and Slug mRNA levels were examined by RT-qPCR. The results are expressed as the mean ± SEM of at least three independent experiments. Values that are statistically different from one another (p < 0.05) are indicated by different letters