Fig 2: RP11-364P22.2 facilitates IL-1ß-induced ATF3 protein expression and nucleus translocation. A, Comparison of ATF3 expression in normal (Control) and OA cartilage tissues. B, qPCR validation of overexpression and knock-down of RP11-364P22.2 in chondrocytes (left), neither of which affected transcription of ATF3 (right). C, Western blot showing depletion of RP11-364P22.2 decreased IL-1ß-induced ATF3 protein expression. D and E, Western blot (D) and immunofluorescence (E) showing IL-1ß-induced nucleus translocation of ATF3 in chondrocytes was abolished upon depletion of RP11-364P22.2. F, Western blot showing depletion of RP11-364P22.2 decreased while ectopic expression of the lncRNA increased the stability of ATF3 protein in chondrocytes. RP11, RP11-364P22.2; KD, knock-down; OE, overexpression

Fig 3: ATF3 is upregulated in Ang II-induced cardiac fibroblasts. (A) mRNA and (B) protein expression levels of ATF3 were assessed after treatment with Ang II by RT-qPCR and western blotting, respectively. (C) mRNA and (D) protein expression levels of ATF3 were assessed after transfection with different plasmids by RT-qPCR and western blotting, respectively. Data are presented as the mean ± SD. *P<0.05, ***P<0.001. ATF3, activating transcription factor 3; Ang II, angiotensin II; Oe, overexpression; NC, negative control; si, small interfering RNA; RT-qPCR, reverse transcription-quantitative PCR.

Fig 4: miR-27a-3p Targets the 3' UTR of ATF3(A) Atf3 transcript level in primarily cultured rVSMCs treated with Pi. The results were obtained from an mRNA microarray (GEO: GSE74755). (B) miR-27a-3p mimic significantly attenuated the luciferase activity driven by Atf3-3' UTR. psiCHECK2-Atf3-3' UTR was used for luciferase activity measurement. (C) miR-27a-3p inhibitor dramatically increased the luciferase activity. (D) miR-27a-3p mimic failed to inhibit mutant Atf3-3' UTR where the miR-27a-3p-binding sequence was altered. (E) miR-27a-3p mimic significantly reduced the mRNA transcript amount of Atf3. (F and G) miR-27a-3p mimic reduced the protein amount of Atf3 as determined by western blot analysis. Quantification results from the western blot (F) are shown in (G). Error bars indicate SD. *p < 0.05, **p < 0.01. NS, not significant.

Fig 5: Overexpression of ATF3 inhibits Ang II-induced viability, migration and fibrosis in human cardiac fibroblasts. (A) Cell viability was evaluated using a Cell Counting Kit-8 assay. (B) Cell migration was assessed using the wound healing assay (magnification, ×100). (C) Protein expression levels of MMP-1 and MMP-2 were semi-quantified using western blotting. (D) Immunofluorescence staining was performed to assess the protein expression levels of a-SMA (magnification, ×200). (E) Protein expression levels of CTGF, fibronectin, collagen I and collagen III were semi-quantified using western blotting. Data are presented as the mean ± SD. *P<0.05, ***P<0.001. ATF3, activating transcription factor 3; Ang II, angiotensin II; a-SMA, a-smooth muscle actin; CTGF, connective tissue growth factor; Oe, overexpression; NC, negative control.