Fig 1: Physically binding of RP11‐364P22.2 to ATF3 is necessary for the catabolic effects of IL‐1β in chondrocytes. A, Time‐dependent MTT assay showing blocking the interaction between RP11‐364P22.2 and ATF3 by antisense (AS) oligo abolished IL‐1β‐caused chondrocytes growth retardation. B, Representative flow cytometry plots showing addition of antisense oligo abolished IL‐1β‐induced apoptosis in chondrocytes. C, Western blot showing the attenuated effects of IL‐1β in synthesis of the extracellular matrix proteins and collagenase 3 as well as activation of NF‐κB in chondrocytes upon blocking RP11‐364P22.2/ATF3 binding by antisense oligo. AS, antisense; NC, negative control
Fig 2: RP11‐364P22.2 facilitates IL‐1β‐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‐1β‐induced ATF3 protein expression. D and E, Western blot (D) and immunofluorescence (E) showing IL‐1β‐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.
Supplier Page from Abcam for Anti-ATF3 antibody [EPR22610-19] - ChIP Grade