Fig 2: RORa regulates IL-6 expression by interacting with IL-6 promoters.A Real-time PCR analysis of TNF-a, IL-1ß, IL-6, and IL-17 mRNA in human chondrocytes infected with RORA knockdown lentivirus (sh-RORA) or control lentivirus (sh-NC) (n = 3). B Schematic diagram of the potential binding site for RORa in the promoter region of IL-6 using JASPAR database. C ChIP-qPCR enrichment assay was performed on chondrocytes with anti-RORa antibody or IgG. The chondrocytes were pretreated with vehicle or 1 µM SR3335 for 6 h. IgG immunoprecipitation was used as a negative control. D IHC staining for IL-6 of articular cartilage after indicated adenovirus injection for 6 weeks in mice. Scale bars, 200 µm for ×100 picture and 50 µm for ×400 picture. E ELISA assay to detect IL-6 protein expression after transfected with RORA overexpression or vector plasmid for 48 h (n = 4). The statistical data in A, C, and E were analyzed with Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, ns = no significance. All data shown above are presented as mean ± SD.

Fig 3: RORα inhibition reversed IL-6 mediated cartilage degradation in human articular chondrocytes.A IHC staining and positive cell percentage for STAT3 and phosphorylated STAT3 (Y705) of normal and degenerated OA cartilage (n = 6). Scale bars, 50 μm. B IHC staining for phosphorylated STAT3 of articular cartilage after 6 weeks of indicated treatment of mice. Scale bars, 50 μm. C Cells were infected with sh-RORA or control virus (sh-NC) lentivirus and then treated with 50 ng/ml IL-6 for 48 h. The expressions of MMP13, ADAMTS4, ADAMTS5, ACAN, and COL2A1 in human articular chondrocytes were detected by real-time PCR analysis. D Western blot analysis and quantification of ACAN, COL2A1, ADAMTS4, ADAMTS5, and MMP13 protein level in human chondrocytes. Chondrocytes were infected with sh-RORA sh-NC lentivirus and then treated with 50 ng/ml IL-6 for 48 h as indicated. E The expression of RORα in human articular chondrocytes after different durations of IL-6 treatment was detected by western blot. F The expressions of MMP13, aggrecan, and COL2A1 in human chondrocytes were detected by western blot. Cells were treated with either vehicle, IL-6, SR3335, Stattic, or in combination as indicated above. The statistical data in A were analyzed with Student’s t test, and data in C and D were analyzed with one-way ANOVA followed by Dunnett’s test. *P < 0.05, **P < 0.01, ns = no significance. All data shown above are presented as mean ± SD.

Fig 4: POU6F1 coordinates with RORA to inhibit LUAD progression.A–D Dual-luciferase assay indicating the relative promoter activity of HIF1A, ENO1, PDK1, and PRKCB in A549 and NCI-H1299 cells transfected with mock, RORA, CRISPRi-Scb, or CRISPRi-POU6F1 #1. E, F Real-time qRT-PCR and western blotting assays showing the expression of POU6F1, RORA, HIF1A, ENO1, PDK1, and PRKCB in A549 cells transfected with mock, RORA, CRISPRi-Scb, or CRISPRi-POU6F1 #1. G, H Representative images (left panel) and quantification (right panel) of soft-agar (G) and transwell (H) assays indicating the growth and invasion of A549 cells transfected with mock, RORA, CRISPRi-Scb, or CRISPRi-POU6F1 #1. Student’s t-test and ANOVA compared the difference in A–E and G–H. *P < 0.05, **P < 0.01.

Fig 5: Postnatal ERß expression completely, while postnatal RORA expression partly, reverses prenatal MPA exposure-mediated OXT suppression and oxidative stress in offspring. The pregnant dams were treated with either control (CTL) or MPA, and the subsequent offspring received either empty (EMP), ERß (?ERß) or RORA (?RORA) lentivirus, and the offspring were then sacrificed for biological assays. (A–F) The hypothalamic tissues from PVN area were isolated for biological assays: (A) mRNA levels, n=4. (B) Protein quantitation, n=5. (C) Representative western blots for (B). (D) OXT levels in hypothalamic tissues, n=5. (E) Superoxide anion release, n=5. (F) 8-OHdG generation, n=5. (G) OXT levels in CSF, n=5. (H) OXT levels in serum, n=5. *P < 0.05, vs. CTL/P-EMP group; ¶ P < 0.05, vs. MPA/P-EMP group. Data were expressed as mean ± SD.