Additional file 3: of Methotrexate upregulates circadian transcriptional factors PAR bZIP to induce apoptosis on rheumatoid arthritis synovial fibroblasts

Plasmid constructs of Per2/Bik promoter. D-box(+), plasmid constructs containing D-box. D-box(−), plasmid constructs without D-box. D-box motifs of Per2 promoter were mutated from 5′-TTATGTAA-3′ to 5′-CGCCAGGC-3′ (−372 to −365), and 5′-TTACGTAA-3′ to 5′-CAGCGTAA-3′ (−47 to −40). Human Bik promoter containing D-box (−780 to +176) and human Bik promoter without D-box (−260 to +323) constructed. (PDF 215 kb

Additional file 1: of Methotrexate upregulates circadian transcriptional factors PAR bZIP to induce apoptosis on rheumatoid arthritis synovial fibroblasts

The interactions of circadian clock genes and its relative factors. BMAL/CLOCK and PER/CRY create the circadian rhythm via E-box, and DBP, TEF, HLF, E4BP4 via D-box. BMAL and CLOCK heterodimerize and bind to E-box elements on promoter regions of Per and Cry genes to induce their transcription. Thereafter, PER and CRY proteins heterodimerize to inhibit activities of their own and other E-box regulated promoters. (PDF 214 kb

Additional file 5: of Methotrexate upregulates circadian transcriptional factors PAR bZIP to induce apoptosis on rheumatoid arthritis synovial fibroblasts

The mRNA expression of circadian clock genes over time. mRNA expression of circadian clock genes measured at –4 h (before synchronization), 0 h (just before MTX stimulation), 24 h, 32 h, and 48 h. Controls and 10/100 nM of MTX showed almost the same expression rhythms, and MTX influenced their expression levels. (PDF 264 kb

Additional file 2: of Methotrexate upregulates circadian transcriptional factors PAR bZIP to induce apoptosis on rheumatoid arthritis synovial fibroblasts

Detailed information of patients with RA enrolled in this study. Joint samples obtained from 10 different patients to establish primary cultured synovial fibroblast cell lines. Age, sex, disease duration, CRP, DAS28-ESR, and treatment with MTX, PSL, and other DMARDs shown in the table. (PDF 252 kb

Additional file 6: of Methotrexate upregulates circadian transcriptional factors PAR bZIP to induce apoptosis on rheumatoid arthritis synovial fibroblasts

Two different transcriptional pathways by which MTX induces apoptosis to synovial fibroblasts: PAR bZIP–Per2 transcriptional pathway and PAR bZIP–Bik transcriptional pathway. We propose MTX induces apoptosis in synovial cells through activated binding of PAR bZIP to D-box in two different genes, Per2 and Bik, and these dual pathways work independently but synergistically. (PDF 206 kb

Additional file 4: of Methotrexate upregulates circadian transcriptional factors PAR bZIP to induce apoptosis on rheumatoid arthritis synovial fibroblasts

Cell viability of MTX-treated fibroblasts. Cell viability of RA synovial fibroblasts measured by WST-8 assay after 24 h of stimulation of MTX (1 pM to 1 μM). MTX (1 and 10 nM) significantly decreased cell viability as shown in Fig. 1, while 1–100 pM of MTX did not. (PDF 274 kb