Figures & data
Figure 1. LncRNA TUG1 silencing promoted autophagy and inhibited inflammation in TGF-β1-exposed RLE-6TN cells.
Note: RLE-6TN cells stimulated with TGF-β1 were transfected with sh-NC or sh-TUG1-1/-2. (a) qRT-PCR determined TUG1 expression in RLE-6TN cells. (b) LncATLAS database analysis of the subcellular localization of TUG1. (c) The subcellular localization of TUG1 in RLE-6TN cells was analysed by FISH assay. (d) CCK-8 assay detected cell viability. (e) Western blotting analysis of autophagy-related protein expression. (f) Immunofluorescent staining for LC3 expression in RLE-6TN cells. (g) The levels of TNF-α, IL-1β, and IL-6 in RLE-6TN cells were assessed by ELISA kits. *, P < 0.05, **, P < 0.01, ***, P < 0.001. Data are from three biological replicates.
Figure 2. LncRNA TUG1 depletion suppressed EMT and fibrinogenesis in TGF-β1-stimulated RLE-6TN cells.
Note: (a) Transwell assay for evaluating the migratory ability. (b) EMT-related protein levels were assessed by Western blotting analysis. (c) Western blotting for determining the levels of myofibroblast markers α-SMA, collagen I and Fibronectin. (d) The protein levels of p-PI3K, PI3K, p-Akt, Akt, p-mTOR, and mTOR were measured by Western blotting. *, P < 0.05, **, P < 0.01, ***, P < 0.001. Data are from three biological replicates.
Figure 3. 3-MA abolished the protection of TUG1 knockdown against TGF-β1-induced inflammation, EMT and fibrosis in RLE-6TN cells.
Note: RLE-6TN cells transfected with sh-NC or sh-TUG1 were treated with autophagy inhibitor (3-MA). (a) qRT-PCR for measuring TUG1 level. (b) Western blotting for detecting the levels of autophagy-related proteins. (c) Immunofluorescent staining for LC3 expressions. (d) The TNF-α, IL-1β, and IL-6 levels were detected by ELISA kits. (e) The migratory potential was detected by transwell assay. (f) Western blotting analysis for assessing EMT-related protein levels. (g) The expression of myofibroblast markers α-SMA, collagen I and Fibronectin was evaluated by Western blotting. (H) The protein levels of p-PI3K, PI3K, p-Akt, Akt, p-mTOR, and mTOR were detected by Western blotting. *, P < 0.05, **, P < 0.01, ***, P < 0.001. Data are from three biological replicates.
Figure 4. LncRNA TUG1 interacted with CDC27 in TGF-β1-exposed RLE-6TN cells.
Note: (a) RIP assay was carried out to validate the binding of TUG1 to CDC27. (b)&(c) Expression of CDC27 in RLE-6TN cells was detected by qRT-PCR and Western blotting. *, P < 0.05, **, P < 0.01, ***, P < 0.001. Data are from three biological replicates.
Figure 5. CDC27 overexpression reversed the effect of lncRNA TUG1 silencing on autophagy and inflammation in TGF-β1-stimulated RLE-6TN cells.
Note: RLE-6TN cells were transfected with Oe-NC or Oe-CDC27 combined with or without sh-TUG1. (a)&(b) The expression of TUG1 and CDC27 was evaluated by qRT-PCR and Western blotting. (c) CCK-8 assay was adopted for cell viability. (d) Western blotting for detecting the levels of LC3-I/II, Beclin-1, and p62. (e) Immunofluorescent staining was performed to evaluate LC3 expression in RLE-6TN cells. (f) TNF-α, IL-1β, and IL-6 levels were determined by ELISA kits. (g) The migration was assessed by transwell assay. *, P < 0.05, **, P < 0.01, ***, P < 0.001. Data are from three biological replicates.
Figure 6. Overexpression of CDC27 counteracted lncRNA TUG1 depletion-mediated inhibition in EMT and fibrinogenesis in TGF-β1-exposed RLE-6TN cells.
Note: (a) The protein levels of E-cadherin, N-cadherin, and Vimentin were assessed by Western blotting analysis. (b) Western blotting for determining the levels of myofibroblast markers α-SMA, collagen I and Fibronectin. (c) The protein levels of p-PI3K, PI3K, p-Akt, Akt, p-mTOR, and mTOR were detected by Western blotting. *, P < 0.05, **, P < 0.01, ***, P < 0.001. Data are from three biological replicates.
Figure 7. Knockdown of lncRNA TUG1 restrained BLM-induced autophagy inhibition and inflammation in rats.
Note: The rats were exposed to BLM and injected with lentiviruses containing sh-NC or sh-TUG1. (a) qRT-PCR detected TUG1 level in the lung tissues of rats, n = 9. (b) HE staining was carried out to observe pathological changes in lung tissues of rats, n = 6. (c) Western blotting for assessing the levels of LC3-I/II, Beclin-1, and p62 in lung tissues, n = 3. (d) ELISA kits were adopted to evaluate TNF-α, IL-1β, and IL-6 levels in lung tissues, n = 3. (e) The protein level of CDC27 was detected by Western blotting, n = 3. *, P < 0.05, **, P < 0.01, ***, P < 0.001.
Figure 8. LncRNA TUG1 silencing suppressed EMT and pulmonary fibrosis in BLM-induced IPF in rats.
Note: (a) The levels of EMT-related proteins in lung tissues were measured by Western blotting analysis, n = 3. (b) Masson’s trichrome staining for evaluating pulmonary fibrosis, n = 6. (c) α-SMA, collagen I, and Fibronectin levels in lung tissues were detected by Western blotting, n = 3. (d) Immumohistochemical staining for α-SMA and collagen I expression in lung tissues, n = 6. (e) The protein levels of p-PI3K, PI3K, p-Akt, Akt, p-mTOR, and mTOR in lung tissues were detected by Western blotting, n = 3. *, P < 0.05, **, P < 0.01, ***, P < 0.001.
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