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. 2021 Jun 4;17(9):2294–2307. doi: 10.7150/ijbs.57915

Figure 2.

Figure 2

CircHIPK3 is involved in TGF-β1-derived fibroblast activation and proliferation. (A) CircHIPK3 and HIPK3 mRNA expression were determined by qRT-PCR in MRC-5 cells treated with 0, 1, 2, 5 ng/ml TGF-β1 for 48h (n = 3), with **P < 0.01 vs. the control group. (B) RT-PCR validated the existence of circHIPK3 in MRC-5 cell lines. CircHIPK3 was amplified by divergent primers in cDNA but not gDNA. GAPDH was used as a negative control. (C-D) The expression of circHIPK3 and HIPK3 mRNA in MRC-5 was detected by RT-PCR or qRT-PCR in the presence or absence of RNase R. (E) The abundance of circHIPK3 and HIPK3 mRNA was assessed by qRT-PCR MRC-5 cells treated with Actinomycin D (2μM) at the indicated time points. (F) Expression of circHIPK3 and HIPK3 in nuclear and cytoplasm of MRC-5 were measured via qRT-PCR analysis. (G) Fluorescence in situ hybridization (FISH) assay was conducted to determine the subcellular localization and expression of circHIPK3 in control and TGF-β1-treated groups. (H) Western blot analysis of Fibronectin, Collagen I, and α-SMA in MRC-5 cells transfected with circHIPK3 siRNA or its negative control then treated with 5ng/ml TGF-β1 for 48h. (I) Immunofluorescence staining of α-SMA in MRC-5 cells for the indicated groups. Red represents α-SMA staining; blue represents nuclear DNA staining by DAPI. (J) EDU assays of MRC-5 cells transfected with control or circHIPK3 siRNA were performed to evaluate cell proliferative ability.