Fig 2: Knockdown and overexpression of lncRNA LURAP1L-AS1 alters the effects of PDGF-BB on fibroblast activation and interaction between LURAP1L and IKKa. hOMF cells were constructed for knocking down and overexpressing LURAP1L-AS1. The fibroblast activation model was established using transfected cells. Expression of lncRNA LURAP1L-AS1 in hOMF cells with LURAP1L-AS1 (A) knocked down and (B) overexpressed. (C) Western blot analysis of the expression of FAP-a, a-SMA, IKKa, I?Ba, NF-?B p65, and p-p65 after knocking down LURAP1L-AS1 and treatment with PDGF-BB. *P<0.05 vs. GV248; ?P<0.05 vs. LURAP1L-AS1-RNAi; ?P<0.05 vs. GV248+PDGF-BB. (D) Western blot analysis of the expression of FAP-a, a-SMA, IKKa, I?Ba, NF-?B p65 and p-p65 after overexpressing LURAP1L-AS1 and treatment with PDGF-BB. *P<0.05 vs. GV146; #P<0.05 vs. LURAP1L-AS1-OE. lncRNA, long non-coding RNA; OE, overexpression; GV248, negative control; GV146, negative control; PDGF, platelet-derived growth factor; hOMF, human oral mucosa (p3) 500K fibroblast; LURAP1L, leucine-rich adaptor protein 1-like; LURAP1L-AS1, LURAP1L antisense RNA 1; p, phosphorylated; a-SMA, a-smooth muscle actin; FAP-a, fibroblast activation protein-a; IKKa, I?B kinase a; I?Ba, nuclear factor of ? light polypeptide gene enhancer in B-cells inhibitor a; RNAi, RNA interference.

Fig 3: Result of the target gene prediction and dual-luciferase reporter assay. The cells were treated with 30 ng/ml PDGF-BB in 10% fetal bovine serum for 72 h for three passages. (A) Reverse transcription-quantitative PCR analysis was performed to evaluate the expression of LURAP1L. Data are presented as the mean ± SEM of triplicate experiments. (B) Western blot analysis of LURAP1L. Densitometry was used to determine the LURAP1L/ß-actin ratios. Data are presented as means ± SEM of three independent experiments. (C) Dual-luciferase reporter assay was performed to verify the regulatory effect of LURAP1L-AS1 on LURAP1L. *P<0.05. LURAP1L-AS1-NC, LURAP1L-AS1 empty plasmid as the negative control; LURAP1L-AS1, vector plasmid expressing target LURAP1L-AS1; 3'UTR-NC: Empty plasmid without 3'UTR-LURAP1L negative control; 3'UTR-LURAP1L, plasmid harboring LURAP1L at the 3'UTR. PDGF, platelet-derived growth factor; LURAP1L, leucine-rich adaptor protein 1-like; LURAP1L-AS1, LURAP1L antisense RNA 1; NC, negative control; UTR, untranslated region; hOMF, human oral mucosa (p3) 500K fibroblast.

Fig 4: Effects of the PDGFR-ß inhibitor, CP-673451 on LURAP1L-AS1-LURAP1L/IKK/I?B/NF-?B signaling and CAF programming. (A) Reverse transcription-quantitative PCR analysis of LURAP1L and LURAP1L-AS1 expression levels. (B) Western blot analysis of FAP-a, a-SMA, IKKa, I?Ba, NF-?B p65 and p-p65 expression following overexpression of LURAP1L-AS1 and treatment with PDGF-BB. *P<0.05 vs. PDGF-BB. PDGF, platelet-derived growth factor; LURAP1L, leucine-rich adaptor protein 1-like; LURAP1L-AS1, LURAP1L antisense RNA 1; IKKa, I?B kinase a; IKK, I-?B kinase; I?Ba, nuclear factor of ? light polypeptide gene enhancer in B-cells inhibitor a; NF-?B, nuclear factor-?B; CAF, cancer-associated fibroblasts; p, phosphorylated; a-SMA, a-smooth muscle actin; FAP-a, fibroblast activation protein-a.

Fig 5: Demonstration of interaction between LURAP1L and IKKa in the NF-?B signaling pathway. (A) Protein-protein interaction analysis in database (www.uniprot.org) (49). (B) Co-immunoprecipitation was used to analyze the interaction between LURAP1L and IKKa. hF, hOMF; hF-BB, hOMF+PDGF-BB; LU-BB, LURAP1L-AS1-RNAi+PDGF-BB; LURAP1L, leucine-rich adaptor protein 1-like; LURAP1L-AS1, LURAP1L antisense RNA 1; IKKa, I?B kinase a.