Fig 1: The regulation of CRLF1 by TGF-ß1 is SMAD3-dependent. (A) CRLF1 expression in the LF cells with or without TGF-ß1 treatment (5 ng/ml). (B) Quantitative analysis of gray values. (C) CRLF1 mRNA expression increased significantly after 24 h of treatment with TGF-ß1 (11.8 ± 2.3-fold change) (n = 3, **p < 0.01). (D) CLCF1 mRNA expression increased after 24 h of treatment with TGF-ß1 (1.49 ± 0.03-fold change) (n = 3, *p < 0.05). (E) Nuclear translocation of SMAD3 by TGF-ß1 (5 ng/ml, 1 h), and inhibition of the translocation by SIS3 (10 µM) in LF cells. (F) SIS3 inhibited the expression of CRLF1 mRNA promoted by TGF-ß1 (5 ng/ml, 24 h) (n = 3, **p < 0.01). Scale bars: 20 µm. The data represent the mean ± SD.
Fig 2: Bipedal standing mice can simulate the pathological changes of human HLF. (A) The AAV2-GFP was successfully transfected into the LF. scale bars: 5 μm. (B) Mouse LF cells transfected with siCRLF1 3# showed a marked reduction of CRLF1 after 24 h of TGF-β1 (5 ng/ml) treatment. (C) The LF area in bipedal standing mice was increased compared to that in control mice. Scale bars: 20 μm. (D) Quantitative analysis of the LF area (n = 8, **p < 0.01). (E) IHC staining of CRLF1 in mice LF. Scale bars: 10 μm. (F) Quantitative analysis of the number of CRLF1-positive cells (n = 8, **p < 0.01). (G) Representative images of EVG-stained LF. Scale bars: 5 μm. (H) Quantitative analyses of the ratio of elastic fibers area to collagen fibers area (n = 8, ***p < 0.001). CON = control group; BS = 10-weeks of bipedal standing group. The data represent the mean ± SD.
Fig 3: Schematic representation of how CRLF1 promotes HLF formation. TGF-β1 can promote the transcription of pro-fibrotic mRNA. CRLF1 signaling is required to regulate the translation of pro-fibrotic factors by activating the ERK pathway. Inhibition of CRLF1 can reduce fibrosis caused by multiple upstream stimuli.
Fig 4: CRLF1 plays a crucial role in LF fibrosis. (A) CRLF1 expression induced by TGF-β1 in LF cells transfected with siRNAs. (B,C) ERK signaling pathway activation by CRLF1 and TGF-β1. (D) siCRLF1 attenuated the ERK phosphorylation induced by TGF-β1. (E) siCRLF1 reduced the fibrosis markers expression induced by TGF-β1. (F,G) Inhibition of the ERK pathway attenuated the CRLF1- and TGF-β1-induced expressions of fibrosis markers in LF cells. (H) siCRLF1 significantly reduced the pro-fibrotic effect of IL-1β (20 ng/ml). (I) siCRLF1 significantly reduced the pro-fibrotic effect of mechanical stretching forces.
Fig 5: CRLF1 plays a crucial role in the formation of HLF in vivo. (A–F) Representative images of H&E-stained LF. Scale bars: 20 µm. (G) Quantitative analyses of the LF area (n = 8, **p < 0.01 versus the AAV-vector group, ##p < 0.01 versus the AAV-vector+BS group). CON = control group; BS = 10-weeks of bipedal standing group. The data represent the mean ± SD.
Supplier Page from Abcam for Anti-CRLF1 antibody